Blood Perfusion and Wound Healing Following Alveolar Bone Regeneration Procedures

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

By

Lamees R. Alssum, B.D.S.

Graduate Program in Dentistry

The Ohio State University

2016

Thesis Committee:

Dr. Binnaz Leblebicioglu, Advisor

Dr. Dimitris Tatakis

Dr. Erdal B. Selnur

Copyright by

Lamees R. Alssum, B.D.S.

2016

Abstract

Objective: This study aims to determine the rate of recovery from surgical trauma through blood perfusion and to evaluate dimensional changes after bone regeneration surgeries.

Material and Methods: Adult patients who are scheduled to receive bone regeneration surgeries in maxillary non-molar single tooth site were recruited.

Clinical parameters including pocket depth (PD), amount of keratinized tissues (KT) and tissue biotype were recorded at baseline and 4 months after the treatment,

Plaque (P) and bleeding on probing (BoP) were recorded at baseline and 3, 6, 9 days, 1 and 4 months. Laser Doppler was used to determine tissue blood perfusion level prior to and immediately after surgery and, at 3, 6, 9 days, 1 and 4 months post operatively. Wound closure was determined through clinical parameters and by using hydrogen peroxide test. Wound healing and possible complications were documented by using specific clinical scales. Wound fluid samples were collected from the wound site by using sterile paper strips. Wound fluid was used to determine the expression of angiogenic markers related to wound healing. CBCT scans were taken immediately after the surgery and at 4 months follow up. Different measurements were obtained including, linear, bucco-lingual (BL) and 3-

Dimensional (3-D) and compared between the 2 data sets.

ii Results: 15 subjects (8 Males, 50 ± 5 years) were recruited. Patients were assigned to 2 groups based on the treatment they received (SP, 9 patients and GBR,

6patients). Healing was uneventful in both groups except for 1 subject (early wound opening). Blood perfusion level dropped immediately after surgery in both groups

(61% and 15% reduction in SP and GBR group, respectively). This was followed by a hyperemic response during early healing period. The recovery rate in the GBR group was slower compared to SP group. Wound fluid volume showed increase level from baseline at 3 days (0.6 ± 0.1 in SP; P<0.01 & 0.7 μl± 0.1 μl in GBR; P<0.01). Only

IL-8 and TGF-α levels were statistically different between the two groups (P<0.05).

Mean linear bone change was - 0.75 ± 0.24 mm in SP and - 0.97 ± 0.16 mm in GBR group. Mean 3-D bone change was – 0.22 ± 0.08 cm3 and – 0.33 ± 0.07 cm3 in SP and

GBR groups, respectively. The difference between the two groups was statistically significant for the 3-D measurements (P=0.05).

Conclusion: Blood perfusion after bone regeneration procedures shows ischemic- reperfusion model. Blood perfusion increased during early healing period and remained elevated relative to baseline levels. GBR showed slower healing compared to SP group, despite higher rate of wound exposure in SP group. Bone resorption can be expected after bone regeneration procedures with more bone remodeling in

GBR group.

iii

Dedication

Dedicated to my Husband, Family and Teachers

iv Acknowledgments

I would like to express my special appreciation and thanks to my advisor Dr.

Binnaz Leblebicioglu for her insight, help and guidance throughout my Masters. I also would like to thank Dr. Dimitris Tatakis for his help and guidance. I also wish to thank Dr. Erdal B. Selnur for his assistance through the CBCT data analysis part of the study. I would like to thank Dr. Tim Eubank for his assistance with the cytokines data in his research laboratory facilities and Mr. Vedat Yildiz for his help in analyzing the data. My sincere thanks also goes to Autumn Gray, Lo-Shen Chen, and

Nicole Scheckelhoff for their hard work and dedication to assist me throughout the project period. Also, I would like to thank all of the Graduate periodontology residents for their support. Lastly, I would like to thank King Saud University for the scholarship. The Division of Periodontology, The Ohio State University-College of

Dentistry, supported this study.

v Vita

2010……………...…..…………Bachelor of dental Sciences, King Saud University, Riyadh,

Saudi Arabia

2011 ……………………………Teaching assistant, King Saud University

2013– 2016……………..……Post-doctoral Training in Periodontics, The Ohio State

University

Peer-reviewed abstracts:

1. Burashed M, Alssum L, Chen L, Scheckelhoff N. Christian L, Tatakis DN, Leblebicioglu B. Treatment and Quality of Life Outcomes Following Guided Bone Regeneration. Abstract #1542. AADR 2014.

2. Alssum L, Chen L, Rotenberg S, Tatakis DN, Leblebicioglu B. Laser Doppler Flowmetry Calibration for Physiological Gingival Blood Flow. Abstract #642. AADR 2014.

3. Alssum L, Gray A, Yildiz V, Tatakis DN, Leblebicioglu B. Retrospective study to determine clinical outcomes of bone regeneration procedures. Abstract #4409. AADR 2015

4. Leblebicioglu B, Burashed M, Alssum L, Gross AC, Eubank TD, Christian L, Yildiz V, Tatakis DN. Pain experience and wound fluid content following guided bone regeneration. Abstract #266, AADR 2015 (oral presentation)

5. Leblebicioglu B, Burashed M, Alssum L, Gross AC, Eubank TD, Christian L, Yildiz V, Tatakis DN. Large Size Ridge Defects: Early Healing Outcomes Following GBR. Abstract#628, EuroPerio 2015 (oral presentation)

vi 6. Alssum L, Erdal B, Yildiz V, Tatakis DN, Leblebicioglu B. Blood Perfusion and Wound Healing Following Socket Preservation/GBR Procedures. oral presentation. Abstract # 264, AADR 2016

7. Scheckelhoff N, Alssum L, Tatakis DN, Leblebicioglu B. Gingival Blood Perfusion Changes Following Non- Surgical Periodontal Therapy. Accepted as poster presentation. Abstract #1943, AADR 2016

8. Gulertekin Z, Alssum L, Gray A, Yildiz V, Tatakis DN, Leblebicioglu B. Retrospective Analysis of Guided Bone Regeneration Failures. Accepted as poster presentation. Abstract #1145, AADR 2016

9. Barriere T, Alssum L, Yildiz V, Tatakis DN, Leblebicioglu B. Peri-implant Treatment Modalities and Outcomes. Accepted as poster presentation. Abstract #1685, AADR 2016

Fields of Study

Major Field: Dentistry

vii TABLE OF CONTENTS

Abstract ………………………………………………………………………………………ii

Dedication...... iv

Acknowledgments...... v

Vita...... vi

List of Figures...... xi

List of Tables...... xiii

Chapter 1: Introduction

• Introduction……………………………………………………………………….1

• References …………………………………………………………………………9

Chapter 2: Guided Bone Regeneration Outcomes In A Training Center-

A Retrospective Study

• Abstract ………………………………………………………………….11

• Introduction...... 12

• Materials and Methods...... 14

• Statistical Analysis...... 16

• Results...... 17

• Discussion...... 19

• Conclusions...... 23

viii • References...... 29

Chapter 3: Blood Perfusion And Wound Healing Following Alveolar Bone Regeneration

Procedure

• Abstract …………………………………………………………………...31

• Introduction...... 33

• Materials and Methods...... 34

• Statistical Analysis...... 41

• Results...... 41

• Discussion...... 49

• Conclusions...... 56

• References...... 69

Chapter 4: CBCT Analysis To Determine Dimensional Ridge Changes Following Bone

Regeneration Surgeries

• Abstract ……………………………………………………………………73

• Introduction...... 74

• Materials and Methods...... 76

• Statistical Analysis...... 80

• Results...... 81

ix • Discussion...... 83

• Conclusions...... 87

• References...... 93

Chapter 5: Conclusion ……………………………………………………….…………….96

Complete references ………………………………………………………………………..97

x List of Figures

Figure.1: flow chart of the data collection process………………………………………………24

Figure. 2: Indications for the corrective surgeries after the first GBR procedures…28

Figure. 3: Site-specific characteristics……………………………………………………...…………28

Figure 4: Plque accumulation (percent) over time……………………………………………..57

Figure 5: Bleeding on probing (percent) over time……………………………………………..57

Figure 6: Amount of collected wound fluid (µl) over time……………………………………58

Figure 7: wound healing- clinical parameters. MWH: mature wound healing, H2O2 positive hydrogen peroxide test (percent)…………………………………………………………..58

Figure 8: visual analogue scale (VAS) for pain analyses over time……………………….59

Figure 9: wound healing clinical parameters. G.mobility: graft mobility………………59

Figure 10: gingival blood perfusion for SP and GBR group over time…………………..60

Figure 11: Angiopoietin -2 concentration in wound fluid over time…………………….63

Figure 12: Endoglin concentration in wound fluid over time………………………………63

Figure13: VEGF-C concentration in wound fluid over time…………………………………64

Figure14: PLGF concentration in wound fluid over time…………………………………….64

Figure 15: EGF concentration in wound fluid over time. ……………………………………65

Figure 16: TGF- α concentration in wound fluid over time…………………………………65

Figure 17: IL-6 concentration in wound fluid over time……………………………………..66

xi Figure 18: IL-8 concentration in wound fluid overtime………………………………………66

Figure 19: IL-18 concentration in wound fluid over time……………………………………67

Figure 20: TNF- α concentration in wound fluid over time…………………………….…..67

Figure 21: Linear measurement……………………………………………………….……………….88

Figure 22: Bucco-lingual (BL) measurement………………………………………………….….83

Figure 23: 3-Dimensional object……………………………………………………………………….88

Figure 24. GBR case (top) at baseline (left) and 4 months follow up (right) and SP case (bottom) at baseline (left) and 4 months follow up (right)………………………….89

Figure 25: comparing SP and GBR in the change in linear and 3-dimensional measurements between baseline and 4 months…………………………………………………90

xii List of Tables

Table .1: data collected for the 143 documented GBR cases…………………………..…25

Table .2: data collected for the 15 documented GBR cases with corrective

surgeries ……………………………………………………………………………………………………….26

Table .3: statistical analysis of the effect of different factors on the need for corrective surgery…………………………………………………………………………………..………27

Table 4: demographics for SP and GBR group……………………………………………....…56

Table 5. Correlation between tissue biotype and LDF readings at baseline and 4 months follow up. ………………………………………………………………………………………...…60

Table 6. Association between LFD readings and wound closure………………………..61

Table 7: concentration of different angiogenic markers in wound fluid overtime61

Table 8. Correlation between angiogenic markers and LDF readings…………………68

Table9 : differences between baseline and 4 months follow up in linear and 3- dimensional measurements………………………………………………………………………………90

Table 10: width of the ridge at 4 months post-operative visit. ………………………90

Table 11: outcome of bone regeneration surgeries……………………………………………..91

Table 12: correlation between LDF data and bone changes…………………………………91

Table 13: correlation between tissue biotype and bone changes at 4 months………91

xiii Table 14: correlation primary wound closure and bone changes at 4 months………92

Table 15: correlation between wound closure and bone changes at 4 months……..92

xiv

CHAPTER 1

INTRODUCTION

Alveolar bone dimensions change throughout the life span and this dynamic

remodeling phenomenon is affected by various factors; Developmental phase is

controlled by teeth formation and eruption. With tooth loss, the alveolar ridge

undergoes atrophy and remodeling causing dimensional reductions both in height

and width. These changes are more pronounced on width of the ridge compared to

ridge height1, 2 and on the buccal than lingual aspect of the ridge3, 4. Related changes

will result in translocation of the diminished ridge to a more palatal or lingual position3,

which can negatively affect outcomes of various treatment modalities used for oral

rehabilitation.

Bone loss after tooth extraction is well documented in the literature, by using

animal models as well as in clinical trials1-4. In an experimental study on dogs,

Araujo and Lindhe4 reported marked dimensional alterations in the first eight

weeks following extraction of mandibular premolars. They concluded that bone

resorption after tooth extraction occurs in two overlapping phases: In phase I,

bundle bone was resorbed and replaced with woven bone, followed by a second

phase of bone resorption on the outer surfaces of the ridge. Since buccal bone

1 crest comprised solely of bundle bone, substantial reduction of the buccal bone height occurs. The relative vertical bone loss on the buccal wall was 2.2 mm, i.e. 45μm/day over 8 weeks observation period. Human studies evaluated ridge changes using different methods including measurements on study casts1-3, radiographs1, 2 and surgical re-entry procedures5. Johnson1 measured the dimensional changes following tooth extraction in the maxilla over 12 months period using study casts and radiographs. He reported that loss of alveolar bone width is more pronounced than height both for anterior and posterior segments.

Pietrokovski and Massler3 reported changes after single tooth extraction measured on 149 casts by comparing the edentulous site with its opposite site of the arch. The buccal plate was resorbed to a greater extend compared to the palatal site of the ridge for both maxillary and mandibular arches. In 2003, another study2 evaluated bone healing and soft tissue contour using study casts and subtraction radiography after single posterior tooth extraction. Over 12 months follow up period, the width of the alveolar ridge reduced by 50% with two third of the changes occurring in the first 3 months. Only slight changes in height compared to the width was reported.

Human re-entry studies showed 3.79 mm (29-63 %) horizontal bone loss and 1.24 mm (11-22%) vertical bone loss 6 months following tooth extraction5. The magnitude of these changes is important to better plan and deliver patient care.

Several surgical techniques and biomaterials have been developed to prevent bony wall loss during extraction and to regenerate already lost bone. The main two procedures invented for these purposes are socket preservation (SP) and guided

2 bone regeneration (GBR). SP is defined as socket augmentation using a variety of bone graft materials with and without membrane barriers at the time of tooth extraction while GBR is defined as the bone augmentation technique with the use of barrier membrane to promote the ingrowth of osteogenic cells and prevent the migration of undesirable cells from the overlying soft tissue into the wound. GBR technique can be applied to extraction defects, ridge augmentation and correction of defects around implants.6 For the purpose of this research project and to avoid confusion, SP will be used to indicate socket grafting without barrier membrane. On the other hand, GBR will always imply the use of barrier membranes. These procedures have been widely tested in controlled and uncontrolled studies with various materials and clinical approaches: bone grafting alone, including autografts, allografts, xenografts, and alloplasts as well as biologic mediators; membrane alone, whether absorbable or not and, membrane in conjunction with bone grafts and mediators7-14.

Early studies by Lekovic and coworkers utilizing non resorbable ePTFE membrane7 and bioabsorbable membrane8 for GBR procedures at the time of extraction showed significant improvement in ridge dimensions compared to control site. A SP technique using a combination of bone graft (FDBA) and collagen membrane was presented few years later by Iasella et al9. Grafted sites lost 1.2 mm in width and gained 1.3 mm in height compared to 2.6 mm and 0.9 mm bone loss in width and height in non grafted extraction sites, respectively. Similar findings were reported with the use of xenograft (corticocancellous porcine bone and collagen

3 membrane) for ridge preservation10. After 7 months of healing, test group showed mean bone loss of 2.5 mm in width and 0.55 mm in height. While control group showed 4.5 mm and 3.3 mm loss in width and height, respectively. Other materials including hydroxyapatite11, bioactive glass12, recombinant human Bone

Morphogenetic Protein-2 (rhBMP-2)13 and medical grade Calcium Sulphate

Hemihydrate (MGCSH)14 have been also used for ridge preservation. Regardless of the material used, ridge preservation techniques although did not prevent bone loss after extraction, result in significantly less vertical and horizontal contraction of alveolar bone crest compared to extraction sites. On average, the mean difference between the treatment and control groups accounted for 1.47 mm and 1.83 mm in height and width, respectively15. The reported variations in these dimensions among various studies may be related to wound healing patterns and host response against chosen biomaterials.

Surgical trauma, including flap elevation, vertical incisions and suturing, can impede the blood circulation in the treated area. This can affect the soft tissue as well as the hard tissue healing. Wood et al16 showed loss of alveolar crest bone height following full and partial thickness apically positioned flap procedures. They stated that the amount of bone loss is closely related to the thickness of the supporting structures, with more bone loss to be anticipated in thin periodontal biotype cases. In a dog model, Fickl et al17 reported more bucco-lingual bone loss after extraction in flapped group compared to flapless group. The additional flap elevation led to 0.7 mm more reduction of the buccal dimensions

4 compared with the flapless procedure. In addition, bone and membrane placement underneath the flap can further interfere with the normal revascularization after surgery. The ability of the periodontal soft tissue to overcome the surgical trauma and temporary ischemia is important for wound healing.

Laser Doppler flowmetry (LDF) is used to measure the tissue blood perfusion noninvasively. It can detect circulatory disturbances at early stages. Baab et al. 18 were the first to report the blood flow of the oral tissue at different locations and under various stimuli. Gingival blood flow was assessed after different periodontal surgeries using LDF readings to evaluate the recovery time from the surgical trauma as well as to evaluate the surgical outcomes. Immediate temporary ischemia after periodontal surgery was reported, with recovery to the baseline levels at 15 days post-operatively19, 20. It also has been shown that flap design can play a role in healing and recovery after surgical trauma. Donos et al20 showed that simplified papilla preservation flap is associated with faster recovery of the post-operative gingival blood flow compared with the modified Widman flap. The applicability of using LDF for monitoring blood perfusion in flaps overlying membrane after GTR procedure was evaluated in an animal model.21 The contralateral site served as control with no membrane. The mean LDF value was lower on the membrane side for each of the 4 observation time points (immediately after surgery, 24, 48, 72 hours). 21

Initial soft tissue thickness may have a differential effect on the rate of

5 blood perfusion recovery. The term ‘‘periodontal biotype’’ was introduced by

Seibert and Lindhe.22 Based on this definition, the gingiva can be classified as “thick- flat” or “thin-scalloped”, with ≤ 1.1 mm gingival thickness generally defined as thin biotype.23 Different studies evaluated the relationship of gingival thickness to the underlying bone thickness. Fu et al.24 measured gingival and bone thickness in cadavers clinically and radiographically using caliper and CBCT scans. They reported an association between labial gingival thickness and underlying bone thickness as measured with CBCT. In another study,25 a modified radiographic technique was used to measure the gingival and alveolar bone thickness on the buccal aspect of the ridge. Parallel profile radiographs were taken with a lead plate positioned on the buccal gingiva to serve as a fixed point for measurements.

Positive correlation was found between the gingival thickness and the bone thickness at different levels.25

Tissue biotypes have been also linked to the outcomes of periodontal and implant therapy. An initial gingival thickness was found to be the most significant factor associated with a complete root-coverage procedure following soft tissue augmentation procedure.26 In immediate single tooth–implant restorations, patients with ‘‘thin-scalloped’’ mucosa often had more tissue recession.27 On the contrary, patients with ‘‘thick-flat’’ mucosa tended to maintain the implant papillae height. 28

These observations suggested that initial soft tissue biotype might be a significant factor influencing regenerative therapy outcomes.

The purpose of the current study is to determine the recovery time and

6 the role of blood perfusion following SP and GBR surgery. The working hypothesis is that there is a difference in blood perfusion recovery between various procedures based on placement of a membrane between flap and host bone, depending on tissue biotype. The ultimate goal of this study is to determine the role of blood supply through soft tissue flap on the outcome of bone regeneration and the fate of buccal plate. The following specific aims were investigated:

1- Changes in blood perfusion following SP/GBR surgery.

2- Early wound healing phases in relation to changes in blood perfusion of the flap covering the wound.

3- Effect of soft tissue biotype on changes in blood perfusion following socket preservation/GBR surgeries.

4- Effect of the rate of blood perfusion on bone fill outcome.

In order to investigate above specific aims, a preliminary retrospective study on GBR and outcomes was initially conducted to determine the frequency of repeated GBR procedures and related complications in our training center

(Chapter II). This was followed with a prospective study, which included SP and

GBR wound sites with similar anatomical locations and similar wound sizes.

Wound healing was documented by using clinical parameters, cone beam computed tomography (CBCT) as well as by collecting wound fluid and studying protein content. This second study included a 4 months follow-up period in order

7 to determine early healing outcomes (Chapters III and IV).

The findings of this study may help better understand the role of blood perfusion on wound healing and bone regeneration and develop treatment modalities to have a better control over outcomes following regenerative procedures.

8 References:

1. Johnson K. A study of the dimensional changes occurring in the maxilla following tooth extraction. Aust Dent J 1969;14:241-244. 2. Schropp L, Wenzel A, Kostopoulos L, Karring T. Bone healing and soft tissue contour changes following single-tooth extraction: a clinical and radiographic 12-month prospective study. Int J Periodontics Restorative Dent 2003;23:313- 323. 3. Pietrokovski J, Massler M. Alveolar ridge resorption following tooth extraction. J Prosthet Dent 1967;17:21-27. 4. Araujo MG, Lindhe J. Dimensional ridge alterations following tooth extraction. An experimental study in the dog. Journal of clinical periodontology 2005;32:212-218. 5. Tan WL, Wong TL, Wong MC, Lang NP. A systematic review of post- extractional alveolar hard and soft tissue dimensional changes in humans. Clin Oral Implants Res 2012;23 Suppl 5:1-21. 6. McAllister BS, Haghighat K. Bone augmentation techniques. Journal of periodontology 2007;78:377-396. 7. Lekovic V, Kenney EB, Weinlaender M, et al. A bone regenerative approach to alveolar ridge maintenance following tooth extraction. Report of 10 cases. Journal of periodontology 1997;68:563-570. 8. Lekovic V, Camargo PM, Klokkevold PR, et al. Preservation of alveolar bone in extraction sockets using bioabsorbable membranes. Journal of periodontology 1998;69:1044-1049. 9. Iasella JM, Greenwell H, Miller RL, et al. Ridge preservation with freeze-dried bone allograft and a collagen membrane compared to extraction alone for implant site development: a clinical and histologic study in humans. Journal of periodontology 2003;74:990-999. 10. Barone A, Aldini NN, Fini M, Giardino R, Calvo Guirado JL, Covani U. Xenograft versus extraction alone for ridge preservation after tooth removal: a clinical and histomorphometric study. Journal of periodontology 2008;79:1370-1377. 11. Hoad-Reddick G, Grant AA, McCord JF. Osseoretention? Comparative assessment of particulate hydroxyapatite inserted beneath immediate dentures. Eur J Prosthodont Restor Dent 1994;3:61-65. 12. Froum S, Cho SC, Rosenberg E, Rohrer M, Tarnow D. Histological comparison of healing extraction sockets implanted with bioactive glass or demineralized freeze-dried bone allograft: a pilot study. Journal of periodontology 2002;73:94-102. 13. Fiorellini JP, Howell TH, Cochran D, et al. Randomized study evaluating recombinant human bone morphogenetic protein-2 for extraction socket augmentation. Journal of periodontology 2005;76:605-613. 14. Aimetti M, Romano F, Griga FB, Godio L. Clinical and histologic healing of human extraction sockets filled with calcium sulfate. Int J Oral Maxillofac Implants 2009;24:902-909.

9 15. Vignoletti F, Matesanz P, Rodrigo D, Figuero E, Martin C, Sanz M. Surgical protocols for ridge preservation after tooth extraction. A systematic review. Clin Oral Implants Res 2012;23 Suppl 5:22-38. 16. Wood DL, Hoag PM, Donnenfeld OW, Rosenfeld LD. Alveolar crest reduction following full and partial thickness flaps. Journal of periodontology 1972;43:141-144. 17. Fickl S, Zuhr O, Wachtel H, Bolz W, Huerzeler M. Tissue alterations after tooth extraction with and without surgical trauma: a volumetric study in the beagle dog. Journal of clinical periodontology 2008;35:356-363. 18. Baab DA, Oberg PA, Holloway GA. Gingival blood flow measured with a laser Doppler flowmeter. Journal of periodontal research 1986;21:73-85. 19. Retzepi M, Tonetti M, Donos N. Comparison of gingival blood flow during healing of simplified papilla preservation and modified Widman flap surgery: a clinical trial using laser Doppler flowmetry. Journal of clinical periodontology 2007;34:903-911. 20. Donos N, D'Aiuto F, Retzepi M, Tonetti M. Evaluation of gingival blood flow by the use of laser Doppler flowmetry following periodontal surgery. A pilot study. Journal of periodontal research 2005;40:129-137. 21. Zanetta-Barbosa D, Klinge B, Svensson H. Laser Doppler flowmetry of blood perfusion in mucoperiosteal flaps covering membranes in bone augmentation and implant procedures. A pilot study in dogs. Clin Oral Implants Res 1993;4:35-38. 22. Seibert JL LJ. Esthetics and periodontal therapy. In: Lindhe J, ed Textbook of Clinical Periodontology 2nd ed Copenhangen, Denmark: Munksgaard; 1989:477-514. 23. Muller HP, Heinecke A, Schaller N, Eger T. Masticatory mucosa in subjects with different periodontal phenotypes. Journal of clinical periodontology 2000;27:621-626. 24. Fu JH, Yeh CY, Chan HL, Tatarakis N, Leong DJ, Wang HL. Tissue biotype and its relation to the underlying bone morphology. Journal of periodontology 2010;81:569-574. 25. Stein JM, Lintel-Hoping N, Hammacher C, Kasaj A, Tamm M, Hanisch O. The gingival biotype: measurement of soft and hard tissue dimensions - a radiographic morphometric study. Journal of clinical periodontology 2013;40:1132-1139. 26. Baldi C, Pini-Prato G, Pagliaro U, et al. Coronally advanced flap procedure for root coverage. Is flap thickness a relevant predictor to achieve root coverage? A 19-case series. J Periodontol 1999;70:1077-1084. 27. Evans CD, Chen ST. Esthetic outcomes of immediate implant placements. Clinical oral implants research 2008;19:73-80. 28. Romeo E, Lops D, Rossi A, Storelli S, Rozza R, Chiapasco M. Surgical and prosthetic management of interproximal region with single-implant restorations: 1-year prospective study. J Periodontol 2008;79:1048-1055.

10

CHAPTER 2

GUIDED BONE REGENERATION OUTCOMES IN A TRAINING CENTER-

A RETROSPECTIVE STUDY

Abstract:

Objectives: Guided Bone Regeneration (GBR) outcome is not predictable when applied to treat large combination type defects. The purpose of this retrospective study was to determine the rate of complications following GBR in a training center.

Materials and Methods: Patients who received GBR prior to dental implant placement and any possible corrective procedure at the same anatomical area within last 5 years were screened by using the scheduling/billing software. The time of implant placement and crown delivery for the same site was documented.

Patient age, gender, anatomical location and surgeon training year were recorded.

The charts of the subjects who had more than one GBR procedure done at the same location were manually screened to determine complications. Statistical analysis using Fisher exact and Chi-square tests performed based on case numbers.

Results: 15 of 143 cases (11%) had secondary corrective surgeries to create sufficient bone to place dental implants (mean age 48±4 yrs; 9 female) in 27 sites.

Most sites with complication were related to area treated by extraction and GBR

(41.5%), large size defect (27.6%) and in the maxillary anterior location (60%).

11 Major complications requiring secondary corrective surgery were lack of implant primary stability (33.3%), infection or delayed wound healing following initial GBR procedure (7 cases, 29.2%) lack of sufficient bone augmentation/graft integration from previous GBR (20.8%) and occurrence of dehiscence at time of implant placement (16.7%). In 3 cases (20%), implant placement was not accomplished despite the repeated procedure.

Conclusions: Within the limits of this study, it appears that the prevalence of GBR failure is low and it is mostly associated with insufficient ridge width and significant dehiscence development at time of implant placement. Anatomical location, size of the defect and type of GBR procedures may be risk predictors for such complications.

Introduction:

Guided bone regeneration (GBR) is based on the principles of guided tissue regeneration, where a barrier membrane is utilized to create a space and selectively allow osteogenic cells to differentiate and proliferate into the wound area promoting new bone formation1. Buser was the first to apply this principle for ridge augmentation in early 1990s2. GBR technique can be applied to extraction defects, deficient alveolar ridges in height and/or width dimensions as well as peri-implant bony defects such as, dehiscence and fenestration. Different barrier membranes, resorbable or non resorbable, have been used in conjunction with bone graft

12 materials (autografts, allografts, alloplasts and xenografts) to maintain the space for bone regeneration.

Success of ridge augmentation techniques can be measured by the amount of regenerated bone and /or the ability to place previously planned dental implants without the need for additional grafting. Overall, regeneration is more predictable for horizontal ridge (width) augmentation compared to vertical

(height) augmentation or combination type (deficient in width and height) of alveolar defects. For horizontal bone augmentation, new bone formation ranges from 1.5-5.5 mm with the use of PTFE membrane after 6-10 months of healing.3 On the other hand, in a systematic review for vertical augmentation techniques, up to 8 mm of vertical bone gain can be achieved with GBR procedure.4 Lang et al.5 measured the amount of bone formation in relation to the available space for regeneration underneath Gore-Tex membrane. They demonstrated that 90-100% of the available space was filled with bone after 6-8 months of undisturbed healing.

Fugazzotto 6 reported success rate of 97% for horizontal augmentation and 92% for vertical augmentation with the use Gore-Tex membrane in conjunction of various non-autogenous bone grafting materials. This was defined as the ability to place dental implants following GBR procedure and healing period.6

In addition to different materials and techniques, the amount of bone formation after GBR procedure depends on the duration of undisturbed healing and the size of the defect to regenerate. Early membrane removal due to exposure or infection resulted in 0-62% bone fill of the space available for regeneration. Small

13 defects (<70 mm3) show almost complete regeneration, while larger defects

(>90mm3) show 90-93% bone fill.5

GBR is a technically demanding procedure and the outcome heavily depends on various phases of wound healing. A wide range of complications have been reported in the literature, ranging between 0-45.5 %.4 These includes infection3, flap dehiscence and membrane exposure3, 6, sublingual edema, dysesthesia and necrosis of the autogenous block graft 7. However, major complications that result in complete failure of the GBR procedures are reported in less than 15% of the interventions. 8 Thus, based on existing evidence, GBR applied to regenerate combination type (bone loss in both horizontal and vertical direction) large size defects (more than one missing tooth) does not have a predictable outcome. The purpose of this retrospective study was to determine the rate and type of complications following GBR procedure in the advanced periodontics training program at The Ohio State University (OSU).

Material and Methods:

Chart screening procedure:

Patients who received GBR prior to dental implant placement and any possible corrective procedure at the same anatomical area were screened by using specific procedure codes and the scheduling/billing software Windent (Carestream

Dental,TX, USA). Cases that received GBR treatment at OSU Graduate Periodontics

Clinics in the last 5 years and with complete necessary documentation were included. Among these cases, specific subjects with a history of secondary

14 corrective surgery at the same anatomical site were chosen; The hard copy of patient charts who had more than one GBR procedure documented at the same location were manually screened to determine complications (Figure 1). The study protocol was reviewed and approved by OSU Internal Review Board (protocol

#2014H0251).

Data collection:

The following information was gathered from specific scheduling/billing software:

• Patients’ demographics including age and gender.

• Anatomical location for the GBR surgery as:

o Upper Posterior (UP)

o Upper Anterior (UA)

o Lower Posterior (LP)

o Lower Anterior (LA)

• Size of the defect (single tooth or multiple adjacent missing teeth area)

• Residents’ training year (1st, 2nd or 3rd year in training)

• GBR procedure was grouped as:

o Group 1: Particulate bone graft and resorbable membrane (G+M)

o Group 2: Extraction, particulate bone graft and resorbable membrane

(Ext+G+M)

o Group 3: Implant, particulate bone graft and resorbable membrane

(I+G+M)

o Group 4: Autogenous block graft (Block)

15 • Duration of the treatment (from the date of the GBR procedure until the

implant placement)

o > 6 months

o ≥ 6 months

o No implant placement date

Outcome of GBR procedure was documented as following:

• Areas that are scheduled for a second bone grafting procedure before or at

the time of implant placement.

• Feasibility to place implant/ implants in the treatment site.

Actual chart screening:

For cases with corrective or second surgery and cases that did not have a documented surgical date for implant placement date scheduled, patients’ charts were manually screened to document:

• Risk assessment, smoking status and history of periodontal disease.

• Reasons for the corrective surgeries.

• Post-operative complications including, swelling, bleeding, infection, open

wound, membrane exposure, premature sutures loss and other less known

complications.

Statistical analysis:

Descriptive statistics followed by Fisher exact test as well as Chi-square test were used to conduct statistical analysis. Differences were accepted as statistically significant at p≤0.05 level.

16 Results:

Procedural information based on data gathered from specific scheduling/billing software:

Procedural code screening by using specific scheduling/billing software revealed total of 451 GBR cases between 2009-2014. Only 143 cases had sufficient documentation to be included for data analysis. 15 out of the 143 cases (11%) needed corrective surgeries (Figure 1).

Table 1 presents demographics related to GBR procedures performed within the last 5 years at the Graduate Periodontics Clinics, OSU. Majority of subjects receiving this procedure were female (58.7 %). The average age was 56.3 ± 1.7 years at the time of the procedure. 52% of the procedures were performed by a 3rd year resident, followed by 2nd (45%) and 1st year (3%) resident, respectively. Bone graft materials together with a collagen membrane was the most commonly utilized technique for GBR (70%). GBR was performed at the time of extraction and implant placement in 14% and 13% of the cases, respectively. Only 4 cases received an autogenous block graft for ridge augmentation. Most of the treated ridge defects was single area (66%) in the posterior mandible sextant (41%). 42% of the cases received implants in the GBR site within 6 months period while 32% have no documented implant placement date (Table 1).

Most of the cases requiring corrective surgeries at the same GBR site were initially diagnosed and treated for tooth extraction in combination with GBR procedure (41.6%) as shown in Table 2. Only 8.9% of the initial surgical sites were

17 performed on an already edentulous ridge. Multiple site-by-site missing teeth defect was more difficult to regenerate with 27.6% of such sites having corrective surgeries. In terms of anatomical location, 60% of corrective surgeries were performed in maxillary anterior sextant. Corrective surgeries resulted in a delay in the treatment in 60% of the cases (8-51 months), and 3 cases (20%) had no documented surgical date for implant placement despite performed corrective surgery. (Table 2)

Actual chart screening:

Chart review of the 15 cases identified 27 sites that received corrective surgeries. All except one subject were non-smokers. Most of the cases had a history of periodontal disease and related treatment (66.7%) (Table 2). Major complications requiring secondary corrective surgery were lack of sufficient bone augmentation or graft integration from previous GBR procedure (5 sites, 20.8%), infection or delayed wound healing following initial GBR procedure (7 cases,

29.2%), occurrence of dehiscence at the time of implant placement (4 sites, 16.7%), and lack of implant primary stability (8 sites, 33.3%). (Figure 2)

Figure 3 shows site-specific characteristics of the GBR procedures. 60% of corrective surgeries were performed in multiple site defects and one third of the sites (37%) required more than 2 corrective surgeries to create sufficient bone volume (37%). Up to 15% of the sites did not have a documented date or surgery for implant placement procedure following the second corrective GBR surgery.

18 Statistical analysis (Table 3) showed no significant effect of gender or resident’s training year on the need for corrective surgeries (p>0.05). The timing of

GBR procedure had a significant effect on the outcome. GBR at the time of extraction was statistically significant predictor for the second corrective surgery (p< 0.01).

Also the size of the defect and anatomical locations (upper anterior sextant compared to other sextant) were associated with additional surgeries (p< 0.01).

Secondary corrective surgeries caused significant delay in treatment time (p< 0.01).

(Table 3)

Discussion:

GBR is a common procedure performed to correct ridge deficiencies in preparation for implant placement. Understanding the outcomes and complications of such procedure is of great importance. The purpose of our study was to report the rate and types of complications in OSU training center. This retrospective study showed that the incidence of corrective surgeries after GBR procedure is low. Type of the GBR procedure, anatomical location and size of the defect may be risk predictors for such complications.

Most of the corrective surgeries were performed at sites in which initial surgery included tooth extraction. Our group recently reported that even

“atraumatic extraction” causes immediate change in ridge dimensions.16 This could be due to the fact that following extraction continuous remodeling and resorption is expected. A prospective study on extraction socket healing9 showed up to 50% width loss after tooth extraction in 6 months with most loss occurring during the

19 first 3 months of healing. Only slight change in ridge height compared to width was reported. In addition, systematic review of human re-entry studies10 reported 29-

63% width loss and 11-22% height loss after 6 months of tooth extraction. Although

GBR procedure at the time of extraction can limit the bone dimensional changes, some bone loss is to be expected. Augmented extraction sockets showed 1.47 mm and 1.83 mm less bone loss in height and width compared to extraction site with no further intervention.11 The difficulty to obtain primary closure after extraction could be another reason for more complications during healing. Although primary closure over the augmented site is desirable, it cannot be achieved in all extraction cases.

That can be due to the large extraction socket area that require advanced flap with some vertical incisions to achieve primary closure or the choice of the surgeon to minimize the trauma in the site and avoid any flap elevation. Simion et al12 showed compromised bone regeneration in sites with membrane exposure when augmenting around implants placed immediately following extraction, using resorbable and non resorbable membranes. They discussed bacterial contamination and early membrane degradation when exposed to oral cavity as possible factors affecting the regeneration in such cases. On the other hand, recent studies report uneventful healing with secondary intension with membranes left exposed intentionally after ridge preservation technique using collagen membranes and different bone graft materials.13-15 Severe bone loss around teeth planned for extraction for either endodontic or periodontal reasons can result in severe ridge deficiency. These situations require more extensive regenerative procedure to

20 correct ridge dimension in height and width. Finally, alveolar bone loss/ fracture during extraction could be another explanation for our result. Although the incident of buccal bone loss after atraumatic surgical technique in the same training center is low (4%)16, other studies reported alveolar bone fracture in 14.5% of the cases.17

Our study identified anatomical location as a risk predictor factor that affect the outcome of GBR procedure, with upper anterior region showing more complications compared to other locations. Maxillary anterior sextant, in addition to being a highly esthetic demanding area, represents some anatomical challenges.

Anterior teeth are prominent with thin buccal plate that can be easily damaged during the extraction process. Nevins et al18 reported up to 5.24 mm height loss after extraction of maxillary anterior teeth. Augmenting such cases with xenograft material resulted in improved ridge height; nevertheless 2.42 mm height loss was evident. Size of the wound is another risk predictor for GBR complications. In the current study, sites with two or more adjacent missing teeth were associated with more repeats and complications compared to single tooth area. A prospective observational study conducted by our research team19 revealed complications in relation to outcomes following GBR performed to treat large size ridge defects. This result is expected and can be explained by the difficulties in treating big defects including, membrane stabilization, creating regeneration space, tension free closure and to maintain primary closure during healing.

Operator experience level can be crucial for the treatment outcome. Although some studies reported that operator experience is important for the outcome of

21 periodontal therapy such as scaling and root planing19, others reported no effect of experience level on implant survival rate. 20, 21 In our study, residents’ training year was not a significant factor for the outcome of the GBR procedure. Most of the GBR procedures were performed by either a second or third year resident with enough experience in basic surgical techniques. In addition, the close supervision of the periodontal faculties could contribute to this finding.

Complications reported after the GBR procedures that indicate corrective surgeries included lack of sufficient bone, dehiscence or lack of implant stability at the time of implant placement and open wound and delayed healing. This finding is in agreement with the complications reported in the literature.2, 22,6 Lack of sufficient bone to place the desired implant size and the development of dehiscence at the time of implant placement was reported by Fugazzotto6 in 14% of the cases.

In all GBR procedures documented in this study, collagen membrane was used.

According to Tal et al22 spontaneous exposure of collagen membrane after GBR procedure is 23-50%. Documented complications after the second GBR surgery are similar to what was reported after the first surgery. Multiple operations in the same area can result in formation of scar tissue that can be difficult to handle and lead to some complication. Also lack of soft tissue in the treatment site can result in repeated complications or failures.

This study has some limitations. First, the study has a retrospective nature that cannot control exposure or outcome and only relay on the documented variables. Second, lack of enough documentation through Windent software. This

22 can result in underestimation of the actual outcomes and complications could be higher than what we are reporting. Finally, the effect of some confounding variables such as systemic health and smoking cannot be investigated due to small number of identifiable cases.

In summary, within the limitation of this retrospective study, it appears that the incidence of corrective surgeries following GBR procedures is low (11%) even in a training center. Risk predictors for the GBR complications include anatomical location, size of the defect and GBR procedure at the time of tooth extraction.

23

Windent software screening

n=451 GBR cases

n=143

cases with enough documentaion

n=15 cases with corrective surgeries

Figure.1: flow chart of the data collection process

24

143 Documented GBR Cases Gender M 59 (57.2+2 yrs old) (41%) F 84 (55.4+1.4 yrs old) (59%) TRAINING 1st 3 (3%) 2nd 64 (45%) YEAR 3rd 75 (52%) GBR G+M* 101 (70%) Ext+G+M* 20 (14%) Procedure I+G+M* 18 (13%) Block 4 (2%) Size of Single 95 (66%) Multiple 29 (20%) Anatomical defect LP 59 (41%) UA 31 (22%) Location UP 26 (18%) LA 8 (6%) Not Reported 19 (13%) Time in < 6 months 60 (42%) ≥6 months 37 (26%) treatment No Implant placement 46 (32%)

date

Table .1: data collected for the 143 documented GBR cases

(G+M): Particulate bone graft and resorbable membrane,

(Ext+G+M) Extraction, particulate bone graft and resorbable

membrane, (I+G+M) Implant, particulate bone graft and

resorbable membrane, (Block) Autogenous block graft

procedures. LP: lower posterior, UA: upper anterior, UP:

upper posterior and LA: lower anterior

25

15 Documented Cases With Repeated GBR Procedure Gender M 6(10.2% of all M; 40%) F 9(10.7 % of all F; 60%) Smoking status Smokers 1 (6.7%) Non smokers 14 (93.3%) History of Treated 10 (66.7%) No perio history 5 (33.3%) periodontitis TRAINING YEAR 2nd 6 (10.9 % of all 2nd; 40%) 3rd 9 (12.3% of all 3rd; 60%) GBR Procedure G+M* 9 (8.9% of all G+M*; 60%) Ext+G+M* 5 (41.6% of all Ext+G+M*; I+G+M* 0 33.3%) Block 1 (25% of all I+G+M*; 6.6%) Size of defect Single 7 (7.4 % of all single; 46.7 %) Multiple 8 (27.6% of all multiple; Anatomical Location LP 6 (4.2% of all LP; 40%) UA 53.3%) 9(6.3% of all UA; 60%) Time in treatment 6 months 3 (20%) > 6 months (8- 9 (60%) No Implants placement 3 (20%) 51 months) date

Table .2: data collected for the 15 documented GBR cases with corrective surgeries (G+M): Particulate bone graft and resorbable membrane, (Ext+G+M) Extraction, particulate bone graft and resorbable membrane, (I+G+M) Implant, particulate bone graft and resorbable membrane, (Block) Autogenous block graft procedures. LP: lower posterior, UA: upper anterior. The first percentage (%) is in proportion to the whole sample (143 cases) and the second is in proportion to the cases with corrective surgeries (15 cases).

26

No Repeat Repeat p-value Gender M 52 6 0.826 F 73 9 Training year 1st 2 0 0.506 2nd 57 6 3rd 65 9 GBR Procedure G+M* 92 9 0.0004* Ext+G+M* 12 5 I+G+M* 18 0 Block 3 1 Size of defect Single 86 7 0.0039* Multiple 20 8 Anatomical LP 51 6 0.001*

Location UA 21 9 Time in < 6 months 55 3 ≥ 6 months 26 91 91 0.001* treatment No implant 44 3 placement date

Table .3: statistical analysis of the effect of different factors on the need for corrective surgery. * Statistical significant (p<0.05) (G+M): Particulate bone graft and resorbable membrane, (Ext+G+M) Extraction, particulate bone graft and resorbable membrane, (I+G+M) Implant, particulate bone graft and resorbable membrane, (Block) Autogenous block graft procedures. LP: lower posterior, UA: upper anterior.

27

Figure. 2: Indications for the corrective surgeries after the first GBR procedures.

Figure. 3: Site-specific characteristics

28

References:

1. Dahlin C, Linde A, Gottlow J, Nyman S. Healing of bone defects by guided tissue regeneration. Plast Reconstr Surg 1988;81:672-676. 2. Buser D, Dula K, Belser U, Hirt HP, Berthold H. Localized ridge augmentation using guided bone regeneration. 1. Surgical procedure in the maxilla. Int J Periodontics Restorative Dent 1993;13:29-45. 3. Buser D, Bragger U, Lang NP, Nyman S. Regeneration and enlargement of jaw bone using guided tissue regeneration. Clin Oral Implants Res 1990;1:22-32. 4. Rocchietta I, Fontana F, Simion M. Clinical outcomes of vertical bone augmentation to enable dental implant placement: a systematic review. Journal of clinical periodontology 2008;35:203-215. 5. Lang NP, Hammerle CH, Bragger U, Lehmann B, Nyman SR. Guided tissue regeneration in jawbone defects prior to implant placement. Clin Oral Implants Res 1994;5:92-97. 6. Fugazzotto PA. Report of 302 consecutive ridge augmentation procedures: technical considerations and clinical results. Int J Oral Maxillofac Implants 1998;13:358-368. 7. Stellingsma K1 BJ, Stegenga B, Meijer HJ, Raghoebar GM. Satisfaction and psychosocial aspects of patients with an extremely resorbed mandible treated with implant-retained overdentures. A prospective, comparative study. Clin Oral Implants Res 2003;14:166-172. 8. Merli M, Migani M, Esposito M. Vertical ridge augmentation with autogenous bone grafts: resorbable barriers supported by ostheosynthesis plates versus titanium-reinforced barriers. A preliminary report of a blinded, randomized controlled clinical trial. Int J Oral Maxillofac Implants 2007;22:373-382. 9. Schropp L, Wenzel A, Kostopoulos L, Karring T. Bone healing and soft tissue contour changes following single-tooth extraction: a clinical and radiographic 12-month prospective study. Int J Periodontics Restorative Dent 2003;23:313- 323. 10. Tan WL, Wong TL, Wong MC, Lang NP. A systematic review of post- extractional alveolar hard and soft tissue dimensional changes in humans. Clin Oral Implants Res 2012;23 Suppl 5:1-21. 11. Vignoletti F, Matesanz P, Rodrigo D, Figuero E, Martin C, Sanz M. Surgical protocols for ridge preservation after tooth extraction. A systematic review. Clin Oral Implants Res 2012;23 Suppl 5:22-38. 12. Simion M, Misitano U, Gionso L, Salvato A. Treatment of dehiscences and fenestrations around dental implants using resorbable and nonresorbable membranes associated with bone autografts: a comparative clinical study. Int J Oral Maxillofac Implants 1997;12:159-167. 13. Barone A, Ricci M, Tonelli P, Santini S, Covani U. Tissue changes of extraction sockets in humans: a comparison of spontaneous healing vs. ridge

29 preservation with secondary soft tissue healing. Clin Oral Implants Res 2013;24:1231-1237. 14. Cardaropoli D, Cardaropoli G. Preservation of the postextraction alveolar ridge: a clinical and histologic study. Int J Periodontics Restorative Dent 2008;28:469-477. 15. Beck TM, Mealey BL. Histologic analysis of healing after tooth extraction with ridge preservation using mineralized human bone allograft. Journal of periodontology 2010;81:1765-1772. 16. Leblebicioglu B, Hegde R, Yildiz VO, Tatakis DN. Immediate effects of tooth extraction on ridge integrity and dimensions. Clin Oral Investig 2015;19:1777-1784. 17. Adeyemo WL, Ladeinde AL, Ogunlewe MO. Influence of trans-operative complications on socket healing following dental extractions. J Contemp Dent Pract 2007;8:52-59. 18. Nevins M, Camelo M, De Paoli S, et al. A study of the fate of the buccal wall of extraction sockets of teeth with prominent roots. Int J Periodontics Restorative Dent 2006;26:19-29. 19. M. Burashed LA, L Chen, N. Scheckelhoff, L. Christian, D. Tatakis, and B., Leblebicioglu. Treatment and Quality of Life Outcomes Following Guided Bone Regeneration J Dent Res 93 2014:Abstract#1542. 20. Brayer WK, Mellonig JT, Dunlap RM, Marinak KW, Carson RE. Scaling and root planing effectiveness: the effect of root surface access and operator experience. Journal of periodontology 1989;60:67-72. 21. Kohavi D, Azran G, Shapira L, Casap N. Retrospective clinical review of dental implants placed in a university training program. J Oral Implantol 2004;30:23-29. 22. Melo MD, Shafie H, Obeid G. Implant survival rates for oral and maxillofacial surgery residents: a retrospective clinical review with analysis of resident level of training on implant survival. J Oral Maxillofac Surg 2006;64:1185- 1189. 23. Tal H, Kozlovsky A, Artzi Z, Nemcovsky CE, Moses O. Long-term bio- degradation of cross-linked and non-cross-linked collagen barriers in human guided bone regeneration. Clin Oral Implants Res 2008;19:295-302.

30

CHAPTER 3

BLOOD PERFUSION AND WOUND HEALING FOLLOWING ALVEOLAR BONE REGENERATION PROCEDURE

Abstract:

Objective: This study aims to determine the rate of recovery from surgical trauma

through blood perfusion following bone regeneration surgery. The working

hypothesis is that the rate of recovery is different between a wound in which

repositioned flap is used and a wound in which biomaterials are introduced

between flap and host bone.

Material and Methods: Adult patients who are scheduled to receive bone

regeneration surgeries in maxillary non-molar single tooth site were recruited.

Clinical parameters including pocket depth (PD), amount of keratinized tissues (KT)

and tissue biotype were recorded at baseline and 4 months after the treatment,

Plaque (P) and bleeding on probing (BoP) were recorded at baseline and 3, 6, 9

days, 1 and 4 months. Laser Doppler was used to determine tissue blood perfusion

level prior to and immediately after surgery and, at 3, 6, 9 days, 1 and 4 months post

operatively. In addition, wound closure was determined through clinical

parameters and by using hydrogen peroxide test. Wound healing and possible

complications were documented by using specific clinical scales. Wound fluid 31 samples were collected from the wound site by using sterile paper strips. In addition, a soft tissue biopsy was obtained during surgery, at 9 days and at 4 months. Wound fluid and soft tissue biopsy were used to determine the expression of angiogenic markers related to wound healing. Data was collected and analyzed for statistical significance.

Results: 15 subjects (8 Males, 50 ± 5 years) were recruited. Patients were assigned to 2 groups based on the treatment they received (SP, 9 patients and GBR,

6patients). Healing was uneventful in both groups except for 1 subject (early wound opening). 47% of the subjects reported pain during the healing period. Blood perfusion level dropped immediately after surgery in both groups (61% and 15% reduction in SP and GBR group, respectively). This was followed by a hyperemic response during early healing period. The recovery rate in the GBR group was slower compared to SP group. Wound fluid volume showed increase level from baseline at 3 days (0.6 ± 0.1 in SP; P<0.01 & 0.7 μl± 0.1 μl in GBR; P<0.01). Only IL-8 and TGF-α levels were statistically different between the two groups (P<0.05).

Conclusion: Blood perfusion after bone regeneration procedures shows ischemic- reperfusion model. Blood perfusion increased during early healing period and remained elevated relative to baseline levels. GBR showed slower healing compared to SP group, despite higher rate of wound exposure in SP group.

32 Introduction:

Post-extraction alveolar ridge dimensional changes are well documented in animal models1 as well as human subjects2-4. Extraction sites lose more width compared to height, and that is more pronounced on the buccal aspect of the ridge.1-

4 Most of the changes occur in the first year, with more than 60% width loss during the first 3 months.3 Therefore, ridge preservation at the time of extraction is utilized to minimize such changes. Different bone graft materials, with or without barrier membrane have been used in clinical trials.5-10 Ridge preservation techniques generally result in acceptable ridge dimensions for implant placement. A systematic review of ridge preservation outcomes11 reported 1.47mm and 1.83 mm less bone change (less bone loss) in height and width, respectively.

Wound healing after ridge preservation can be accelerated or delayed depending on the material used. The effect at the cellular and molecular level is dependent on the morphology, chemical composition, porosity and particle size of the materials.12 On the other hand, surgical trauma including flap elevation, vertical incisions and suturing may impede the blood circulation to the surgical site. In addition, bone and membrane placement underneath the flap can further interfere with the healing associated revascularization after the surgery. The ability of the periodontal soft tissue to overcome the surgical trauma and temporary ischemia is important for wound healing.

Tissue biotypes have been also linked to the outcomes of periodontal and implant therapy. The initial gingival thickness is reported as to be the most

33 significant factor associated with a complete root-coverage procedure.13 In immediate single tooth–implant restorations, patients with ‘‘thin-scalloped’’ mucosa often had more tissue recession.14 On the contrary, patients with ‘‘thick-flat’’ mucosa tend to maintain the implant papillae height. 15 These observations suggest that initial soft tissue biotype may be a significant factor influencing regenerative therapy outcomes.

Blood perfusion of the surgical site can be monitored using Laser Doppler

Flowmetry (LDF). LDF is non-invasive technique widely used in medicine especially in the field of plastic surgery16, 17. It can detect vascular disturbances at an early stage allowing further intervention and correction. Baab et al. 18 were the first to report the blood flow of the oral tissue at different locations and under various stimuli. Gingival blood flow was assessed after different periodontal surgeries using

LDF readings to evaluate the recovery time from the surgical trauma as well as to evaluate the surgical outcomes.19, 20

The purpose of the current study is to evaluate changes in blood perfusion following bone regeneration surgeries, in relation to early phases of wound healing and soft tissue biotype. Two types of bone regeneration surgery protocols (socket preservation and guided bone regeneration) were compared.

Materials and Method:

Study design:

The study was a prospective observational clinical trial. Patients treatment

34 planned for extraction and/or bone regeneration before implant placement for a single non-molar maxillary site were recruited. Clinical examination and sampling were conducted prior to surgery and at 3, 6, 9 days, 1 month and 4 months post-surgery. Clinical parameters, wound healing measures, GCF sample,

LDF readings and soft tissue biopsy were obtained at different time points. The

Institutional Review Board of The Ohio State University (OSU) approved the study protocol (protocol #2014H0150), data collection and informed consent forms.

Subject population:

Patient referred to the Graduate Periodontology Clinics at OSU for bone regeneration surgeries before implant placement in a single non-molar maxillary site were recruited. Inclusion criteria includes:

• Age between 18 and 65 years old

• Single non molar maxillary tooth site

• Stable periodontal condition

• Good systemic health

Exclusion criteria:

• Systemic condition that can affect wound healing

• Smoking

• Pregnancy

Signed informed consent was obtained from all subjects.

35 Clinical measures:

The patients’ clinical data was collected at baseline and repeated at 3, 6, 9 days, 1 and 4 months post-operatively. Photographic documentation was done at each visit. The following clinical parameters were recorded:

• Pocket depth (PD) for the two adjacent teeth to the treatment site

recorded at baseline and 4 months visit.

• Amount of keratinized tissue (KT) measured on the mid buccal of the

treatment site recorded at baseline and 4 months visit.

• Tissue biotype (TB) obtained by measuring the thickness of the soft tissue

on the treatment site. A non-tension wax caliper used on mid buccal flap

after elevation and 2-3 mm apical to flap edge. Tissue biotype were

classified as thick if the tissue thickness was > 1 mm, and thin if it is ≤ 1

mm (modified from Muller et al.21). This measurement was obtained at

the time of bone regeneration and repeated at implant placement surgery.

• Plaque (P) and Bleeding on probing (BoP) percentages of the treatment

sextant were recorded at baseline, 3, 6, 9 days, 1 month and 4 months,

postoperatively.

• Gingival Crevicular Fluid (GCF) was obtained from adjacent teeth at baseline

and from edges of the wound area at each of the follow up visits by using a

sterile paper strips (Periopaper®). A total of 6 GCF samples were taken at

each time point for 30 seconds sampling time per strip. GCF volume was

determined by using a previously calibrated electronic volume quantification

36 unit (Periotron 8000®). Samples were stored in sterile vials and were

transferred and stored at -20 C for future multiplex assays to determine

angiogenesis markers content within wound fluid.

• Soft tissue biopsies were taken from the treatment site at the time of bone

regeneration and implant placement surgeries. In addition punch biopsy

(3 mm) was taken from the palatal area of the treatment site at 9 days

postoperatively. Samples were immediately frozen in liquid nitrogen and

then stored in -20 C for future analysis through laser capture

microdissection, mRNA extraction and Q-PCR experiments to detect

differential expression of specific angiogenesis related genes during

wound healing.

LDF measurements:

An alginate impression was obtained at baseline visit and stone cast was produced to prepare a template (Stent). The stent was fabricated with a 0.06” thermo-formed material. A small hole created on the mid buccal of the treatment site 3-4 mm from the crest. A plastic sleeve inserted into the hole and was used to stabilize the LDF probe during readings. Thus, LDF probe was held at a standardized position perpendicular to the tissue and at a distance of 0.5-1 mm from the gingiva and remained fixed during repetitive LDF measurements.

Periflux System 5000 PF 5010 LDPM unit (Perimed AB, Sweden) equipped with a standard probe was chosen for LDF measurements. The instrument was calibrated by means of the Perimed PF 1000 Motility Standards

37 according to the manufacture’s specifications prior to actual measurements. The signals were recorded in arbitrary Perfusion Unit (PU) and monitored using the

Perisoft software (PSW 2, version 2.5.5, Perimed AB). During LDF readings, the subjects were comfortably seated and relaxed in an upright position on the dental chair. These measurements were obtained before surgery, immediately after surgery and at 3, 6, 9 days, 1 and 4 months postoperatively. Each reading was recorded for 120 seconds. All LDF measurements were performed by the same examiner.

Surgical procedures:

Surgical procedures were performed by residents of the Advanced

Periodontics Training program at OSU, under direct supervision by periodontal faculty. Patients were assigned into two groups based on the treatment they received:

• After tooth extraction, if the socket walls were intact (four-wall residual

defect), socket preservation (SP) was performed with the use of allograft

bone material (FDBA, Straumann, Andover, MA, USA) and collagen wound

dressing (Collagen Plug, Zimmer Dental, Carlsbad, CA, USA), (SP group)

• If patients presented with ridge deficiency immediately following

extraction (the socket lost one or more walls) or if the site was edentulous,

guided bone regeneration (GBR) was performed with the use of allograft

bone material (FDBA, Straumann, Andover, MA, USA) and resorbable

collagen membrane (Biomend Extend, Zimmer Dental, Carlsbad, CA, USA),

38 (GBR group).

After local anesthesia, atraumatic extraction performed and socket debridement and irrigation were performed. Surgical site inspected to either perform SP or GBR. In case of SP, limited flap elevation performed on the mid buccal of the site and the socket was then filled with FDBA and covered by

Collagen Plug. Sutures placed to secure the Collagen Plug in place. No attempt was made to obtain primary closure and the site was left to heal by secondary intension. While in GBR group, performed at the time of extraction or later, a full mucoperiosteal flap reflected with or without vertical incision. FDBA was placed on the site and covered by resorbable collage membrane; flap was release to obtain tension free primary closure when possible. Based on standard clinical protocol, patients received prescription for antibiotics (5-7 days), analgesics (3-

10 days) and 0.12% chlorhexidine rinse (3 times /day for 2 weeks).

Clinical wound healing:

Visual analogue scale (VAS) was used to monitor patients’ pain and discomfort after the procedure. Clinical wound healing was evaluated at each of the post-operative visits. Clinical wound healing scores (0=Mature wound healing, 1=Erythema, 2=Bleeding, 3=Graft mobility, 4=Suppuration, 5=Necrosis) were used for this purpose (modified from Kloostra et al.22). Each category was given either a score of 0 if absent or a score of 1 if present. Mature wound healing was defined as complete wound closure with no other significant findings/complications. Erythema was defined as increase of redness compared

39 to adjacent non-operated sites. Bleeding was given a score of 1 whenever spontaneous bleeding was detected at the wound site. Graft mobility was evaluated by gentle palpation and evaluation of any loose sub-gingival material by using a periodontal probe. Suppuration was evaluated by detection of the presence or absence of pus or discharge at the wound site. Any visual soft and/or hard tissue necrosis detected was given a score of 1.

Clinical wound closure was documented immediately after surgery and during post-operative visits through clinical observation. In addition, hydrogen peroxide (HP) test was used to determine complete wound closure.23 The amount of any possible wound exposure was measured with a periodontal probe by selecting the largest exposure area and measuring the farthest distance from the two flap margins.

Preparation of wound fluid samples and multiplex assays:

Extraction of crevicular fluid from paper strips was accomplished. First, PCR microtubes were prepared by creating small holes at the bottom of each then they were sterilized. These tubes were placed into sterile 2 ml Eppendorf tubes.A sterile scissor was used to separate the paper strips from the waxed part of the strips.

Then, they were transferred from cryovials into PCR microtubes, soaked in 200 μl cold sterile PBS, and incubated on ice for 15 minutes with occasional vortexing.

Samples then were centrifuged at 13,000 rpm for 10 min. Supernatant was collected from the bottom of the tubes. Approximately 160 μl elution volume was recovered from each tube. The Bio-Plex Pro Human Cancer Biomarker panel 2 was used to

40 detect the presence and the amount of several angiogenesis markers within crevicular fluid samples. The panel included the following cytokines, chemokines and growth factors: Angiopoietin-2, sCD40L, EGF, Endoglin, sFASL, HB-EGF, IGFBP-

1, IL-6, IL-8, IL-18, PAI-1, PLGF, TGF-α, TNF- α, uPA, VEGF-A, VEGF-C and VEGF-D.

Data management and statistical analysis:

Clinical measurements were performed by a single trained examiner, using UNC-15 probe. Data was presented as mean ± standard error. LDF readings were calculated as an average of the 2 minutes period of each individual recording by the Periosoft computer program (PSW 2, version 2.5.5, Perimed AB).

Changes of blood flow values were expressed as the percent difference (ΔPU%) between the PU value at a specific site at a specific observation time point (PUt) and the individual baseline vale of the same site (PU0): ΔPU%= PUt - PU0/ PU0 ×

10019, 24

The data were analyzed using both GraphPad Prism 5 (GraphPad Software, Inc. CA,

USA) and Statistical Analysis Software, version 9.3 (SAS Institute Inc., Cary, NC,

USA). Repeated measure mixed model was used to compare baseline with each time points. Unpaired t-test or Mann-Whitney U test were used to compare SP and GBR group. Q-PCR data was analyzed by using unpaired t-test. Pearson correlation coefficients were calculated to reveal the association between various parameters.

Differences were accepted as statistically significant at p≤0.05 level.

Results:

20 subjects were recruited and 15 completed the study (8 Males, 50 ± 5

41 years). 5 subjects dropped from the study for different reasons (1 subject chose to withdraw after the 1st visit, 2 subjects decided to delay implant placement procedure, LDF stent was not stable for another subject and he dropped from the study and, surgical protocol had to be modified for one subject which made him non-eligible for the study). Each subject contributed a single site resulting in 15 total surgical sites. 9 patients received SP and 6 received GBR surgery. 3 patients received GBR at the time of extraction, while the other 3 had GBR done at 5 weeks, 5 and 8 months after the extraction. Most of the treated sites were localized in maxillary anterior sextant (80%). No statistically significant difference between the two groups in age, gender or anatomic location (P=0.15,

0.83 and 0.64, respectively). Table 4 show the demographics for SP and GBR groups.

Clinical measures:

Clinical parameters (n=15) are presented in Table 4. Mean probing depths

(PD) at the adjacent teeth was 2.3 ± 0.4 mm in SP group and 1.9 ± 0.4 mm in GBR group before treatment, and 2.3 ± 0.4 mm in SP group and 2.1 ± 0.5 mm following

4 months of healing. Mean keratinized tissue (KT) was 5.8 ± 1.3 mm in SP and 5.8

± 0.9 mm in GBR prior to surgery and 5.3 ± 1.5 mm in SP and 4.8 ± 1.2 mm in GBR at 4 months follow-up appointment, respectively. 53.3 % (n = 8) of the sites were classified as having thick tissue biotype. No difference were detected between groups in PD, KT or tissue biotype (Table 4). Plaque and Bleeding on Probing

(BoP) percentages during entire observation time are reported in Figures 4 and 5.

42 The amount of detected plaque decreased from baseline (38 ± 9% in SP & 42 ±

9% in GBR) during early post-operative period and return to baseline level at 1 month follow-up (42 ± 9% in SP & 43 ± 11% in GBR) and decreased again for both groups at the 4 months post-operative visit (29 ± 5% in SP & 37 ± 11% in

GBR). The difference in plaque percent was significant between baseline and 9 days in SP group (P=0.02), and between 9 days and 1 month in both groups

(P<0.01 in SP and P=0.05 in GBR). No significant difference detected between the two groups at any time point (Figure 4). BoP percentage was different between the two groups; In SP group, the BoP decreased from baseline to 3 days follow up

(24 ± 5% to 9 ± 3%) then increased at 6 days (22 ± 6%) followed by a decreased again up to 4 months follow up (10 ± 3% at 4 months), while in GBR group it was almost at the same level up to 9 days followed by an increase thereafter (15 ± 4% at baseline, 15 ± 6% at 3 days, 17 ± 2% at 6 days, 14 ± 4% at 9 days, 20 ± 3% at 1 month and 25 ± 7% at 4 months follow up). The difference in BoP was significant between baseline and 3 days, 9 days and 4 months in SP group (P<0.05). No detectable difference was noted within GBR group. However, between group difference was statistical significant at 4 months follow up (P=0.03) (Figure 5).

The amount of wound fluid collected during healing time is presented in

Figure 6. At baseline, wound fluid volume was comparable in both groups (0.2 ±

0.05 μl for SP and 0.3 ± 0.07 μl for GBR group). Wound fluid increased during early healing time (0.6 ± 0.1 in SP; P<0.01 & 0.7 μl± 0.1 μl in GBR; P<0.01 at 3 days), followed by decrease to baseline levels at 4 months post-operative visit in

43 SP group (0.2 ± 0.02 μl) and in GBR group (0.3 ± 0.07 μl). No significant difference was noted between the groups at any time point (Figure 6).

Clinical wound healing:

No primary closure was attained in SP group at the end of the surgery, while in only 3 subjects (50 %) of the GBR group primary closure was achieved

(Figure 7). No primary closure seen in SP group until 1 month, where all sites achieved primary closure by 1 month in GBR group (33% of the sites showed primary closure at 3 days, 50% at 6 and 9 days, respectively). The difference in wound closure between groups is statistically significant at 3, 6 and 9 days post- operatively (P<0.05). Similarly, 72% of the sites presented open wound with HP test at one month in SP group compared to 20% in GBR group. Only one site

(11%) presented open wound at 4 months follow up in SP group (Figure 7). All subject had un-eventful healing except one (subject#9, GBR group), who experienced early membrane degradation and graft loss. 40 % of the patient reported no pain immediately after the treatment, while 47 % reported pain with different severity. No patient reported pain or discomfort at 1 or 4 months follow up. The difference in pain experience between the two groups was negligible

(Figure 8). Erythema at the wound site observed in all subjects in both groups during 3 and 6 days post-operative visits. Erythema was noted in none of SP sites and 17% of GBR sites by 4 months (Figure 9). Bleeding from the surgical site decreased overtime in both groups. 56 % of the SP and 100% of the GBR sites had bleeding at 3 days, and no bleeding detected at 4 months for both groups (Figure

44 9). No graft mobility detected in SP group during the follow up period. Graft mobility was noted in 17-33% (n=3 subjects) of the GBR group during the first 2 weeks (Figure 9). Necrosis at the wound margin noted on SP and GBR groups in

11% (n=3 subjects) and 67% (n= 5 subjects) of cases during 3 days follow-up.

The difference between groups was statistically significant (P=0.01). No necrosis detected past the 9 days post operative visit in either group (Figure 9).

LDF measurements:

Figure 10 shows the gingival blood perfusion in SP and GBR at different time intervals. At baseline, and before local anesthesia, LDF value was 150 ± 82

PU in SP and 90 ± 38 PU in GBR group. Overall, the blood flow decreased immediately following the surgical procedure in both groups (61 ± 19 % and 15

±69% reduction in SP and GBR groups, respectively). The difference was not statistically significant in either group (P>0.05). At the 3 day post-operative visit, an increase in blood flow can be seen in both groups compared to baseline levels

(111 ±64% and 226 ± 108% increase in SP and GBR groups respectively). At 6 days follow up, the blood flow values dropped to 109 ± 24% in SP group and remained at almost the same level for up to 4 months (106±83%), while in GBR group, the blood flow increased to 286 ± 160%. The blood flow levels remained at a higher level relative to baseline in GBR group at the following post-operative visits (160 ± 68% at 4 months) and, it showed more fluctuation over time. The difference between the baseline level and day 3, 6 and 9 as well as 1 month was significant in GBR group (P<0.05). No significant difference from baseline level

45 can be detected at any time point in SP group (P>0.05). In addition, no significant difference between the two groups at any time points (P>0.05) (Figure 10).

Analysis of differences in LDF readings between tissue biotype shows no differences between the two groups (P=0.72) (Table 5). Analysis of wound closure in relation to LDF readings shows statistical significant association between wound exposure and increased blood perfusion (p≤ 0.01). (Table 6)

Wound fluid angiogenic mediators:

All 18 angiogenesis markers investigated as part of wound healing were detectable within wound fluid samples obtained from either wound or adjacent teeth. Related complete data is presented in Table 7 as mean±s.e. concentration

(pg/ml or ng/ml).

Angiopoitin-2 (Ang-2) level was not detectable in GBR group and was low (2

± 2 ng/ml) in SP group at the baseline. An increase in Ang-2 concentration noted at

3, 6 and 9 days post-operative visits in SP (23 ± 5 ng/ml, 42 ± 9 ng/ml and 46 ± 14 ng/ml, respectively) and at 3 and 6 days post-operative visits in GBR group (27 ±7 ng/ml and 53 ±16 ng/ml, respectively). The difference from baseline level was statistically significant at 6 and 9 days in SP group (P<0.01) and at 6 days in GBR group (P=0.02). These concentrations dropped to the baseline level at 1 month follow up (Figure 11).

Endoglin concentrations were comparable in SP and GBR groups at baseline.

An increase in the concentration of Endoglin was noted at 9 days in SP group (22 ± 6

46 ng/ml) (Figure 12) However, difference from baseline was not statistically significant.

Vascular Endothelial Growth Factor-C (VEGF-C) concentration increased from the baseline values (20 ±4 ng/ml in SP & 30±6 ng/ml in GBR) up to 9 days in

SP group (52 ± 9 ng/ml) and up to 6 days in GBR group (51 ± 5 ng/ml). These concentrations decreased at 30 days and were comparable between the two groups at 4 months follow-up (Figure 13). There was statistical significant difference between the baseline level and 3, 6, 9days and 4 months values in SP group

(P<0.05). On the other hand, the difference from baseline level did not reach statistical significance in GBR group. A sharp increase in the Placental growth factor (PLGF) concentration was seen at 3 days in both groups (6725 ± 125 pg/ml in

SP and 7414±140 pg/ml in GBR) compared to baseline (1821±237 pg/ml in SP and

1417± 246 pg/ml in GBR) and that was statistically significant (P<0.01). PLGF concentration decreased thereafter, but remained elevated compared to baseline levels up to 4 months (Figue 14). The difference from baseline level was still significant at 6 and 9 days follow up in SP and at 6 days in GBR (P<0.01).

Interestingly, Epidermal Growth Factor (EGF) and Transforming growth

Factor- α (TGF- α) (Figures 15 & 16) decreased from the baseline level at 3 days in both groups (EGF, from 21 ± 3 ng/ml to 9 ±1 ng/ml in SP group and from 17 ±3 ng/ml to 8 ±2 ng/ml in GBR group and TGF- α, from 9270±2287 pg/ml to

5513±2159 pg/ml in SP and from 10268±1775 pg/ml to 2973±652 pg/ml in GBR group). The decrease in EGF level was significant in SP group at 3, 6 and 9 days

47 follow up (P≤0.01) (Figure 15). The concentrations increased gradually and peaked at 1 month for GBR group and at 4 months for SP group (Fig.15,16). Only TGF- α concentration was significantly increased at 1 month in GBR group (P=0.01).

Comparing both groups, the difference in TGF- α level was statistically significant at

1-month follow up (P=0.01).

Interlukin-6 (IL-6) concentration increased gradually and reached the highest level at 6 days in GBR group (131 ± 48 ng/ml) and at 9 days in SP group

(145± 64 ng/ml). The difference was significant at 6 days in both groups and at 9 days in SP group (P≤0.01).The levels dropped back to the baseline values at 1 month for both groups (Figure 17).

A sharp increase in Interlukin-8 (IL-8) level noted, which peaks at 3 days in

GBR group (2297 ± 346 ng/ml) and 6 days in SP group (3915 ± 804 ng/ml).

Statistical difference noted at 3,6 and 9 days in SP(P<0.01) and at 3 and 6 days in

GBR (P<0.05). Similar to IL-6, IL-8 concentrations return to baseline levels at 1 month follow-up. There was statistical significant difference between the SP and

GBR groups at 9 days (P<0.01) (Figure 18).

Interlukin-18 (IL-18) concentration shows a sharp decrease in GBR group at

3 days (from 185± 85 ng/ml at baseline to 44± 12 ng/ml at 3 days) and then slowly increases over time to reach baseline level at 4 month follow-up. The differences from baseline level in GBR group was significant at all time points except 4 months

(P<0.05). While in SP group, a gradual decrease noted up to 9 days (from 142±52

48 ng/ml at baseline to 36±8 ng/ml at 9 days; P=0.05) followed by a sudden increase at

1 month visit (195±65 ng/ml) (Figure19).

In SP group, Tumor Necrosis Factor (TNF-α) reaches its highest concentration at 6 days (5536± 963 pg/ml; P=0.01) and return to baseline level at 1 month (2704±508 pg/ml), without statistically significant differences. In contrast,

GBR group showed the highest concentration of TNF-α at 1 month follow-up

(3758±1295 pg/ml) but that also did not reach statistical significance (Figure 20).

Correlation analysis between different angiogenic markers and LDF readings reveals no statistically significant correlation between the two variables (Table 8).

Discussion:

The outcome of wound healing and bone regeneration surgeries is dependent on multiple factors. Age, gender, systemic health, smoking and pregnancy have been reported to affect the wound healing on the patient level.34-36

On the wound level, location and size of the wound, plaque control, surgical technique and biomaterial of choice use can either accelerate or delay the healing process.8, 34, 37, 38 The effect of blood flow and flap revascularization after bone regeneration surgeries have not been investigated. Therefore, it was the purpose of this study to evaluate the changes in gingival blood perfusion after SP and GBR surgeries, and to assess any possible effect on wound healing measures or outcomes.

In a previous retrospective study (chapter II) we identified the maxillary anterior area, GBR at the time of tooth extraction and wound size as possible

49 determinants for GBR outcomes. We designed the current study to focus on bone regeneration surgeries on maxillary single rooted teeth in order to identify any possible factors that can justify our previous observation. In addition, we limited our sample to single tooth area since large size defect can be associated with more extensive surgical techniques and related trauma. Only patients under 65 years old, with good systemic health were included to eliminate any effect on wound healing process. Also smokers were excluded from the study due to the detrimental effect of smoking on healing.

Bone regeneration surgeries aim to preserve or reconstruct the alveolar ridge after tooth loss. Two treatment modalities (e.g. SP and GBR) were compared since they are commonly performed before implant placement. Although they differ in the surgical technique and materials, they are used to achieve the same purpose.

In this study, flap elevation limited to extraction site was performed in SP group in order to make the groups more comparable. After the surgery, no primary closure achieved in SP group, while it was attained in 3 patients in GBR group. In those patients, the surgeon chose to wait at least 6 weeks after tooth extraction for the soft tissue healing before performing the GBR procedure. Thus, primary closure in such cases can be easily achieved. Although some studies showed detrimental effect of open wound on the regenerative outcomes,39 others report no negative effect.40-42

Gingival blood perfusion was monitored using LDF before the surgery and during follow up visits. We reported low blood perfusion immediately after the surgery, which indicates ischemia. This is in accordance with previous studies. 19, 20,

50 43 The finding can be explained by the severance of blood supply during flap elevation or the use of local anesthesia with vasoconstriction.44 Flap ischemia noted in all except 3 patients where increased blood perfusion was noted immediately after the surgery. Local anesthetic type, amount and technique may explain this finding, as well as the duration of the surgery. According to previous studies,20, 43 the effect of vasoconstrictor can last for at least 2 hours. In our study, some of the surgeries lasted longer than 2 hours, which allow drug to metabolize and may explain the hyperemic response seen immediately after the surgery. At 3 days follow up, an increase in blood perfusion was noted, compared to baseline, in both groups.

The blood perfusion showed further increase in GBR group at 6 days follow up and stayed at a higher level compared to baseline in SP group. Pervious studies demonstrated similar findings,19, 20, 43 with increased blood flow following periodontal access surgeries up to 7 days post-operatively. However, the measurements were taken at different anatomical locations, i.e. alveolar mucosa and buccal and palatal papillae. In another study,20 blood perfusion level was compared following two periodontal surgeries, Modified Widman Flap (MWF) and simplified papilla preservation flap (SPPF). They concluded that SPPF were associated with a faster recovery with blood perfusion level back to pre-operative level at 4 days, compared to persisted hyperemia up to 7 days in MWF group. In our study, the blood perfusion level sustained higher levels compared to baseline up to 4 months follow up in both groups, with more fluctuation over time in GBR group. This finding could be explained by the introduction of new materials underneath the flap

51 including bone graft alone or in conjunction with membrane, which can affect the re-establishment of new vasculature at the gingival periosteal level. In addition, although both groups showed similar pattern during early blood flow levels, GBR group showed further increase at 6 days compared to decrease in SP group. At 1 month follow-up, the blood perfusion level was still high in GBR group compared to

SP. This finding may reflect the less traumatic surgical technique in SP, where only limited flap reflection performed, which may allow faster formation and organization of granulation tissues. Furthermore, it is not clear what is the effect of using collagen membrane in re-vascularization of the flap overlying the site.

According to the company product manual (Biomend Extend, Zimmer Dental,

Carlsbad, CA, USA), the used collagen membrane can take up to 18 weeks to resorb and could be present during the entire evaluation period of this study. Finally, changes in the position of the tissues after the surgery (coronally positioned flap) especially in GBR group can make it difficult to find the exact same position of the probe in the subsequent visits . This could be a possible source of variation in LDF readings.

Our analysis showed no difference between thin and thick biotype in blood perfusion level at baseline or 4 months follow up. This finding could be due to the limited penetration of the LDF light into the tissue. A previous study45 reported that

LDF light penetrate only 0.6 mm into the tissue. Thus, regardless how thick the tissue is, it will reflected the blood perfusion on the very superficial layer.

52 Wound fluid volume increased during early healing time, as expected, and returned back to baseline levels at the end of the study period. Different angiogenic markers analyzed in this study. Only the ones that show significant diffrences were presented and will be discussed on the following section. Angiopoietins (Ang-1 through -4) are essentially involved in maturation, stabilization, and remodeling of vessels and they use associated receptor tyrosine kinase Tie-2. Ang-2, representing a natural Tie-2 antagonist which is highly induced at sites of vascular remodeling.25

Endoglin (also known as CD105) is a TGF- β type III auxiliary receptor. Its activation is induced through hypoxia and TGF- β. Endoglin expression found to be elevated during alterations in vascular structure as they occur during embryogenesis, inflammation, and wound healing.26 VEGF is an endothelial cell mitogen, chemotactic agent and inducer of vascular permeability. It exists in 5 isoforms,

VEGF-A, VEGF-B, VEGF-C, VEGF-D and PLGF.27 EGF family includes EGF, TGF-α and heparin binding EGF-like growth factor (HB-EGF). Two structurally related peptides,

EGF and TGF-α, play important roles in the natural mechanism of wound healing.

EGF like peptides are released in wounds as soon as blood clotting occurs, and then during the inflammatory phase, TGF- α is released in the wound area by macrophages. They are angiogenic and chemotactic factors.28 IL-6 can be considered as a pro-inflammatory cytokine that stimulates C-reactive protein and neutrophil elastase (MMP-9) secretion, or anti-inflammatory cytokine by indirectly causing the release of cytokines such as interleukin-10 (IL-10) and interleukin- (IL-4).29 IL-6 has crucial roles in wound healing, probably by regulating leukocyte infiltration,

53 angiogenesis, and collagen accumulation.30 IL-8 is a pro-inflammatory, leukocyte chemo-attractant protein. IL-8 is present as an important specific PMN attractant during the inflammation phase in wound healing.31 IL-18 is a cytokine with many pro-inflammatory functions. Recent studies demonstrated that IL-18 is a potent angiogenic stimulus, with the capacity to mediate vascular migration and new blood vessel formation in the development of inflammatory and angiogenesis-driven disease, such as Rheumatoid Arthritis .32 TNF-α is a pro-inflammatory cytokine that stimulate bone resorption and protease production by fibroblasts and osteoblasts.33

Generally, angiogenesis markers showed an increased concentration during early healing period in both groups. Although most of the markers studied demonstrated similar pattern, there was few exceptions. Ang-2 level was similar in both groups and comparable to the reported pattern in the literature for wound healing.25

However, where the level of Ang-2 dropped after 3 days in GBR group, it sustained high levels in SP group up to 6 days. However, the difference between the groups was not significant. Endoglin showed more robust increased concentration in SP compared to GBR group, but this was not significant. Our finding with VEGF-C is comparable to what is reported in the literature for skin wound healing with high levels between 3 and 7 days.27 However, VEGF-C level was high up to 9 days in SP group. Failla et al.45 reported elevated levels of PLFG between 3-5 days in vivo after full thickness skin wound, which is in agreement with our findings. On the other hand, they reported no detectable values at 7 days after injury, while PLFG had detectable values up to 4 months. Interestingly, EGF, TGF- α and IL-18 showed

54 decreased levels during early wound healing and increased at 1 and 4 months follow-up visits. This could be related to the interaction of the different angiogenic markers, where certain markers over expression lead to suppression of the others.

No clear explanation of this finding can be proposed at this time. The presence of theses angiogenic markers for extended period of time can indicate delayed wound healing. However, existing literature is on skin wound model and comparison with oral wounds could not be applicable.

There are some limitations for this study. First, the sample size is small which can limit the significance of our findings. Second, LDF readings present the change in the flux of blood cells multiplied by velocity. It is calculated as a mean value for each recording over 120 seconds in PU. This number cannot be used as an absolute value due to wide variation between the subjects in blood flow levels. Data was presented as the percentage change relative to baseline level in each subject overtime as suggested by previous studies19, 24. It is not clear if this is the best way to analyze such data.

In conclusion, gingival blood perfusion showed a hyperemic response during early healing period. Blood perfusion level sustained higher levels after bone regeneration surgeries. Blood perfusion showed more fluctuation and slower recovery in GBR group compared to SP group. Only IL-8 and TGF- α show statistical significant difference between the two groups. No correlation was detected between angiogenic markers and blood perfusion level. Blood perfusion is affected by wound

55 closure. The difference in blood perfusion between thin and thick biotype was negligible.

SP (n=9) GBR (n=6) P-value

Age (years ± SD) 54.7 ± 14.8 41.5 ± 18.9 0.15

Gender Male 5 (55.6%) 3 (50%) 0.83

Female 4 (44.4%) 3 (50%)

Anatomic Anterior 7 (77.8%) 5 (83.3%) 0.64

location Premolar 2 (22.2%) 1(16.7%)

PD (mm) Pre-treatment 2.3 ± 0.5 2.3 ± 0.4 0.92

Post-treatment 1.9 ± 0.4 2.1 ± 0.5 0.57

KT (mm) Pre-treatment 5.8 ± 1.3 5.3 ± 1.5 0.55

Post-treatment 5.8 ± 0.9 4.8 ± 1.2 0.10

Tissue Thin 5 2 0.61

biotype Thick 4 4

Table 4: demographics for SP and GBR groups

56

Plaque Accumulation

60

50 + 40 30 * 20 Percentage 10 0 BL 3 DAYS 6 DAYS 9 DAYS 30 DAYS 120 DAYS

SP GBR

Figure 4: Plque accumulation (percent) over time. * SP (BL-9days) P=0.02 + SP and GBR (9days-1 month) P≤ 0.05

BoP * 40 + 30 * * 20

10 percentage 0 BL 3 DAYS 6 DAYS 9 DAYS 30 DAYS 120 DAYS

SP GBR

Figure 5: Bleeding on probing (percent) over time. * SP (difference from the baseline) P <0.05 + SP vs. GBR (P=0.05)

57

Wound Fluid

1 * 0.8 0.6 0.4

0.2 0 ul/30 sec (pooled) BL 3 DAYS 6 DAYS 9 DAYS 30 DAYS 120 DAYS

SP GBR

Figure 6: Amount of collected wound fluid (µl) over time. * SP and GBR (BL-3days) p<0.01

* * *

Figure 7: wound healing- clinical parameters. MWH: mature wound healing, H2O2: positive hydrogen peroxide test (percent) * SP vs. GBR (P<0.05)

58

VAS

3 DAYS 6 DAYS 9 DAYS 30 DAYS 120 DAYS

100 80 60 40 20 PERCENTAGE 0

Figure 8: visual analogue scale (VAS) for pain analyses over time

* *

Figure 9: wound healing clinical parameters. G.mobility: graft mobility. * SP vs. GBR (P=0.01)

59

Gingival Blood Perfusion 500 * 400 * * 300 * 200 100 0 DPU (percentage) -100 BL SX 3 DAYS 6 DAYS 9 DAYS 30 DAYS 120 DAYS SP GBR

Figure 10: gingival blood perfusion for SP and GBR group over time. BL: baseline, SX: after surgery, DPU: difference in perfusion unit * GBR (difference from baseline) P<0.05

Tissue biotype P-value

Thin (n=7) Thick (n=8)

ΔPU% 42 ± 116 77 ± 184 0.666

Table 5. Correlation between tissue biotype and LDF readings at baseline and 4 months follow up.

60

ΔPU% P-value

Primary closure (at the end of the Yes 31 ± 147 < 0.01*

Sergery) No 154 ± 232

Wound Exposure Yes 149 ± 227 < 0.01*

No 43 ± 171

H2O2 test Positive 162 ± 222 0.01*

Negative 73 ± 201

Table 6. Association between LFD readings and wound closure. arker Baseline 3 DAYS 6 DAYS 9 DAYS 30 DAYS 120 DAYS Angiopoeitin-2 (ng/ml) 2±2 23±5 42±9 46±14 0±0 10±0 SP 0±0 27±7 53±16 27±10 4±4 7±5 GBR Hu Endoglin (ng/ml) 10±2 13±3 15±3 22±6 15±4 14±2 SP 12±2 11±1 18±2 15±3 15±2 14±3

GBR VEGFA (ng/ml) SP 237±34 167±31 226±38 183±23 231±35 269±42 181±45 132±29 226±43 152±27 217±42 216±24 GBR VEGFC (ng/ml) SP 20±4 36±6 49±7 52±9 33±7 38±7 30±6 36±5 51±5 45±11 25±5 38±5 GBR VEGFD (ng/ml) SP 66±19 44±9 61±12 55±9 86±15 88±15 69±16 38±7 42±7 53±5 103±25 96±23 GBR PLGF (pg/ml) SP 1821±237 6725±1256 6573±1106 5643±1181 2330±557 2524±911 1417±246 7417±1406 5783±609 4069±847 2610±433 2521±718 GBR Hu EGF (ng/ml) SP 21±3 9±1 10±1 13±2 23±3 24±4 17±3 8±2 9±2 11±2 23±5 20±5 GBR Table 7: concentration of different angiogenic markers in wound fluid over time. Continued

61

Table 7 continued

TGF-α (pg/ml) SP 9270±2287 5513±2159 8537±3404 5717±897 11354±2472 12865±1923 GBR 10268±1775 2973±652 4884±598 8176±1129 15728±3418 13488±2247

HB-EGF (ng/ml) SP 7±1 10±4 11±2 10±2 6±1 13±4 GBR 7±1 5±1 7±1 8±2 9±2 8±1 IL-6 (ng/ml) SP 2±1 44±14 129±47 145±64 2±1 3±1 GBR 1±0 46±8 131±48 71±39 4±2 9±1 IL-8 (ng/ml) SP 573±157 2820±521 3915±804 3738±865 1226±540 1021±234 367±132 2297±346 2162±667 1388±511 526±190 645±149 GBR IL-18 (ng/ml) SP 142±52 106±65 45±7 36±8 195±65 212±72 185±85 44±12 60±13 48±11 103±32 182±93 GBR TNF-α (pg/ml) SP 2863±362 4689±1973 5536±963 4184±1465 2704±508 3267±746 1717±409 2140±382 2421±531 2434±456 3758±1295 3296±811 GBR IGFBL1 (ng/ml) SP 181±98 160±77 235±149 256±86 111±37 284±113 GBR 83±23 90±25 205±60 220±59 130±36 131±67 PAl1(pg/ml) SP 1066±235 3082±622 4993±911 5677±1300 1750±354 1951±444 GBR 802±175 2797±323 5331±841 2980±700 1994±636 1568±406 sCD40L (ng/ml) SP 7±2 12±3 17±3 21±5 9±1 14±5 7±2 10±2 16±1 14±5 9±2 9±2 GBR sFASL (ng/ml) SP 30±5 15±3 19±3 6±7 36±6 38±8 25±5 15±2 17±2 16±4 32±7 19±4 GBR uPA (ng/ml) SP 206±40 207±72 292±79 269±46 241±49 234±55 GBR 182±45 208±45 372±69 255±59 231±57 223±29

Table 7: concentration of different angiogenic markers in wound fluid over time.

62

Angiopoietin-2 80 + * 60 *

40

20

ng/ml pooled in 30 sec 0 BL 3 DAYS 6 DAYS 9 DAYS 30 DAYS 120 DAYS SP GBR

Figure 11: Angiopoietin -2 concentration in wound fluid over time. * SP (difference from baseline) P<0.01 + GBR (BL-6days) P=0.02

Hu-Endoglin 30 25 20 15 10 5 ng/ml pooled in 30 sec 0 BL 3 DAYS 6 DAYS 9 DAYS 30 DAYS 120 DAYS SP GBR

Figure 12: Endoglin concentration in wound fluid over time

63 VEGF-C 70 * 60 * * 50 * 40 30 20

ng/ml pooled in 30 sec 10 0 BL 3 DAYS 6 DAYS 9 DAYS 30 DAYS 120 DAYS SP GBR

Figure13: VEGF-C concentration in wound fluid over time. • SP (difference from the baseline) P<0.05

PLGF * 10000 * 8000 + 6000

4000

2000 pg/ml pooled in 30 sec

0 BL 3 DAYS 6 DAYS 9 DAYS 30 DAYS 120 DAYS SP GBR

Figure14: PLGF concentration in wound fluid over time. * SP and GBR (difference from baseline) P<0.01 + SP (difference from baseline) P<0.01 64

HuEGF 30 25 * 20 * * 15 10 5 ng/ml pooled in 30 sec 0 BL 3 DAYS 6 DAYS 9 DAYS 30 DAYS 120 DAYS SP GBR

Figure 15: EGF concentration in wound fluid over time. * SP (difference from baseline) P≤0.01

TGF-α 25000 * + 20000

15000

10000

pg/ml pooled in 30 sec 5000

0 BL 3 DAYS 6 DAYS 9 DAYS 30 DAYS 120 DAYS SP GBR

Figure 16: TGF- α concentration in wound fluid over time * GBR (difference from baseline) P=0.01 + SP vs. GBR (P=0.01)

65

IL-6 250 + 200 * 150 100 50 0 BL 3 DAYS 6 DAYS 9 DAYS 30 DAYS 120 DAYS ng/ml pooled in 30 sec SP GBR

Figure 17: IL-6 concentration in wound fluid over time * SP and GBR (difference from baseline) P≤0.01 + SP (difference from baseline) P≤0.01

IL-8 Δ 5000 + + * * 4000 * 3000

2000

1000 ng/ml pooled in 30 sec 0 BL 3 DAYS 6 DAYS 9 DAYS 30 DAYS 120 DAYS SP GBR

Figure 18: IL-8 concentration in wound fluid overtime * SP (difference from baseline) P<0.01 + GBR (difference from baseline) P<0.05

66 Δ SP vs.GBR (P<0.01)

IL-18 300 250 + * 200 + + 150 * * 100 * 50

ng/ml pooled in 30 sec 0 BL 3 DAYS 6 DAYS 9 DAYS 30 DAYS 120 DAYS

SP GBR

Figure 19: IL-18 concentration in wound fluid over time * GBR (difference from baseline) P<0.05 + SP (difference from baseline) P =0.05

TNF-α 7000 6000 5000 4000 3000 2000 1000 pg/ml pooled in 30 sec 0 BL 3 DAYS 6 DAYS 9 DAYS 30 DAYS 120 DAYS SP GBR

Figure 20: TNF- α concentration in wound fluid over time

67

ΔPU% Angiogenic markers r P-value

Angiopoietin-2 0.026 0.79

IL-6 0.064 0.52

IL-8 0.088 0.37

IL-18 -0.115 0.24

EGF 0.028 0.86

VEGF-C 0.050 0.61

TGF-a 0.025 0.80

PLGF 0.049 0.62

Table 8. Correlation between angiogenic markers and LDF readings.

68 References:

1. Araujo MG, Lindhe J. Dimensional ridge alterations following tooth extraction. An experimental study in the dog. Journal of clinical periodontology 2005;32:212-218. 2. Johnson K. A study of the dimensional changes occurring in the maxilla following tooth extraction. Aust Dent J 1969;14:241-244. 3. Schropp L, Wenzel A, Kostopoulos L, Karring T. Bone healing and soft tissue contour changes following single-tooth extraction: a clinical and radiographic 12-month prospective study. Int J Periodontics Restorative Dent 2003;23:313- 323. 4. Pietrokovski J, Massler M. Alveolar ridge resorption following tooth extraction. J Prosthet Dent 1967;17:21-27. 5. Aimetti M, Romano F, Griga FB, Godio L. Clinical and histologic healing of human extraction sockets filled with calcium sulfate. Int J Oral Maxillofac Implants 2009;24:902-909. 6. Barone A, Aldini NN, Fini M, Giardino R, Calvo Guirado JL, Covani U. Xenograft versus extraction alone for ridge preservation after tooth removal: a clinical and histomorphometric study. Journal of periodontology 2008;79:1370-1377. 7. Hoad-Reddick G, Grant AA, McCord JF. Osseoretention? Comparative assessment of particulate hydroxyapatite inserted beneath immediate dentures. Eur J Prosthodont Restor Dent 1994;3:61-65. 8. Iasella JM, Greenwell H, Miller RL, et al. Ridge preservation with freeze-dried bone allograft and a collagen membrane compared to extraction alone for implant site development: a clinical and histologic study in humans. Journal of periodontology 2003;74:990-999. 9. Lekovic V, Camargo PM, Klokkevold PR, et al. Preservation of alveolar bone in extraction sockets using bioabsorbable membranes. Journal of periodontology 1998;69:1044-1049. 10. Lekovic V, Kenney EB, Weinlaender M, et al. A bone regenerative approach to alveolar ridge maintenance following tooth extraction. Report of 10 cases. Journal of periodontology 1997;68:563-570. 11. Vignoletti F, Matesanz P, Rodrigo D, Figuero E, Martin C, Sanz M. Surgical protocols for ridge preservation after tooth extraction. A systematic review. Clin Oral Implants Res 2012;23 Suppl 5:22-38. 12. Morjaria KR, Wilson R, Palmer RM. Bone healing after tooth extraction with or without an intervention: a systematic review of randomized controlled trials. Clin Implant Dent Relat Res 2014;16:1-20. 13. Baldi C, Pini-Prato G, Pagliaro U, et al. Coronally advanced flap procedure for root coverage. Is flap thickness a relevant predictor to achieve root coverage? A 19-case series. J Periodontol 1999;70:1077-1084. 14. Evans CD, Chen ST. Esthetic outcomes of immediate implant placements. Clinical oral implants research 2008;19:73-80.

69 15. Romeo E, Lops D, Rossi A, Storelli S, Rozza R, Chiapasco M. Surgical and prosthetic management of interproximal region with single-implant restorations: 1-year prospective study. J Periodontol 2008;79:1048-1055. 16. Svensson H, Pettersson H, Svedman P. Laser Doppler flowmetry and laser photometry for monitoring free flaps. Scand J Plast Reconstr Surg 1985;19:245-249. 17. Yuen JC, Feng Z. Monitoring free flaps using the laser Doppler flowmeter: five-year experience. Plast Reconstr Surg 2000;105:55-61. 18. Baab DA, Oberg PA, Holloway GA. Gingival blood flow measured with a laser Doppler flowmeter. Journal of periodontal research 1986;21:73-85. 19. Donos N, D'Aiuto F, Retzepi M, Tonetti M. Evaluation of gingival blood flow by the use of laser Doppler flowmetry following periodontal surgery. A pilot study. Journal of periodontal research 2005;40:129-137. 20. Retzepi M, Tonetti M, Donos N. Comparison of gingival blood flow during healing of simplified papilla preservation and modified Widman flap surgery: a clinical trial using laser Doppler flowmetry. Journal of clinical periodontology 2007;34:903-911. 21. Muller HP, Heinecke A, Schaller N, Eger T. Masticatory mucosa in subjects with different periodontal phenotypes. Journal of clinical periodontology 2000;27:621-626. 22. Kloostra PW, Eber RM, Wang HL, Inglehart MR. Surgical versus non-surgical periodontal treatment: psychosocial factors and treatment outcomes. Journal of periodontology 2006;77:1253-1260. 23. Marucha PT, Kiecolt-Glaser JK, Favagehi M. Mucosal wound healing is impaired by examination stress. Psychosom Med 1998;60:362-365. 24. Zanetta-Barbosa D, Klinge B, Svensson H. Laser Doppler flowmetry of blood perfusion in mucoperiosteal flaps covering membranes in bone augmentation and implant procedures. A pilot study in dogs. Clin Oral Implants Res 1993;4:35-38. 25. Kampfer H, Pfeilschifter J, Frank S. Expressional regulation of angiopoietin-1 and -2 and the tie-1 and -2 receptor tyrosine kinases during cutaneous wound healing: a comparative study of normal and impaired repair. Laboratory investigation; a journal of technical methods and pathology 2001;81:361-373. 26. ten Dijke P, Goumans MJ, Pardali E. Endoglin in angiogenesis and vascular diseases. Angiogenesis 2008;11:79-89. 27. Bao P, Kodra A, Tomic-Canic M, Golinko MS, Ehrlich HP, Brem H. The role of vascular endothelial growth factor in wound healing. J Surg Res 2009;153:347-358. 28. Schultz G, Rotatori DS, Clark W. EGF and TGF-alpha in wound healing and repair. J Cell Biochem 1991;45:346-352. 29. Okada H, Murakami S. Cytokine expression in periodontal health and disease. Crit Rev Oral Biol Med 1998;9:248-266.

70 30. Lin ZQ, Kondo T, Ishida Y, Takayasu T, Mukaida N. Essential involvement of IL-6 in the skin wound-healing process as evidenced by delayed wound healing in IL-6-deficient mice. J Leukoc Biol 2003;73:713-721. 31. Rennekampff HO, Hansbrough JF, Kiessig V, Dore C, Sticherling M, Schroder JM. Bioactive interleukin-8 is expressed in wounds and enhances wound healing. J Surg Res 2000;93:41-54. 32. Park CC, Morel JC, Amin MA, Connors MA, Harlow LA, Koch AE. Evidence of IL-18 as a novel angiogenic mediator. J Immunol 2001;167:1644-1653. 33. Desborough JP. The stress response to trauma and surgery. Br J Anaesth 2000;85:109-117. 34. Garrett S. Periodontal regeneration around natural teeth. Ann Periodontol 1996;1:621-666. 35. Engeland CG, Bosch JA, Cacioppo JT, Marucha PT. Mucosal wound healing: the roles of age and sex. Arch Surg 2006;141:1193-1197; discussion 1198. 36. Lindhe J, Branemark PI. The effects of sex hormones on vascularization of granulation tissue. Journal of periodontal research 1968;3:6-11. 37. Machtei EE, Cho MI, Dunford R, Norderyd J, Zambon JJ, Genco RJ. Clinical, microbiological, and histological factors which influence the success of regenerative periodontal therapy. Journal of periodontology 1994;65:154- 161. 38. Leblebicioglu B, Salas M, Ort Y, et al. Determinants of alveolar ridge preservation differ by anatomic location. Journal of clinical periodontology 2013;40:387-395. 39. Simion M, Misitano U, Gionso L, Salvato A. Treatment of dehiscences and fenestrations around dental implants using resorbable and nonresorbable membranes associated with bone autografts: a comparative clinical study. Int J Oral Maxillofac Implants 1997;12:159-167. 40. Barone A, Ricci M, Tonelli P, Santini S, Covani U. Tissue changes of extraction sockets in humans: a comparison of spontaneous healing vs. ridge preservation with secondary soft tissue healing. Clin Oral Implants Res 2013;24:1231-1237. 41. Beck TM, Mealey BL. Histologic analysis of healing after tooth extraction with ridge preservation using mineralized human bone allograft. Journal of periodontology 2010;81:1765-1772. 42. Cardaropoli D, Cardaropoli G. Preservation of the postextraction alveolar ridge: a clinical and histologic study. Int J Periodontics Restorative Dent 2008;28:469-477. 43. Retzepi M, Tonetti M, Donos N. Gingival blood flow changes following periodontal access flap surgery using laser Doppler flowmetry. Journal of clinical periodontology 2007;34:437-443. 44. Ahn J, Pogrel MA. The effects of 2% lidocaine with 1:100,000 epinephrine on pulpal and gingival blood flow. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:197-202.

71 45. Failla CM, Odorisio T, Cianfarani F, Schietroma C, Puddu P, Zambruno G. Placenta growth factor is induced in human keratinocytes during wound healing. J Invest Dermatol 2000;115:388-395.

72

Chapter 4

CBCT ANALYSIS TO DETERMINE DIMENSIONAL RIDGE CHANGES FOLLOWING BONE REGENERATION SURGERIES

Abstract:

Objective: Cone beam Computed Tomography (CBCT) can provide a useful tool for evaluation of outcomes of bone regeneration therapy. The aim of this study was to evaluate dimensional changes after Socket preservation (SP) and guided bone regeneration surgeries (GBR).

Materials and Methods: Adult patients who were scheduled to receive bone regeneration surgeries in maxillary non-molar single tooth site were recruited.

Clinical parameters, Laser Doppler flowmetry (LDF) and wound healing were documented at 3, 6, 9 days, 1 and 4 months follow-up appointments (details in chapter III). In addition, CBCT scans were taken immediately after the surgery and at 4 months follow up. Different measurements were obtained including, linear, bucco-lingual (BL) and 3-Dimensional (3-D) and compared between the 2 data sets.

The need for additional bone augmentation at the time of implant surgery was documented.

Results: 15 subjects (8 Males, 50 ± 5 years) were recruited. Patients were assigned to 2 groups based on the treatment they received (SP, 9 patients and GBR,

73 6patients). Mean linear bone change was - 0.75 ± 0.24 mm in SP and - 0.97 ± 0.16 mm in GBR group. Mean 3-D bone change was – 0.22 ± 0.08 cm3 and – 0.33 ± 0.07 cm3 in SP and GBR groups, respectively. The difference between the two groups was statistically significant for the 3-D measurements (P=0.05).

Conclusion: Within the limit of this study, bone resorption can be expected after bone regeneration procedures with more bone remodeling in GBR group.

Introduction:

Bone regeneration surgeries are widely used for preservation and augmentation of alveolar ridges after tooth loss. These can be performed with the use of different bone graft materials with or without barrier membrane or other biological mediators.1-8 A systematic review and Meta analysis9 of the outcomes of bone regeneration surgeries after tooth extraction compared to simple extraction protocol report 1.47 mm and 1.83 mm gain in height and width, respectively. The ridge dimensional changes were determined by using clinical parameters as well as radiographic analysis or surgical re-entry protocols in the presence/absence of guide in order to establish standardization.

Cone beam Computed Tomography (CBCT) technique introduces an innovation in modes of image scanning and volumetric reconstruction of computed tomography (CT) data. This technique has the advantage of relatively low acquisition time and patient radiation dose,10 which make it comparable to traditional dental imaging modalities.11 CBCT has been used in different fields in dentistry including dento-alveolar pathology, oral and maxillofacial surgery,

74 orthodontics, implantology, endodontics, periodontics and general dentistry.10 In periodontics and implant dentistry, it can be utilized for diagnosis,12 treatment planning13 and evaluating outcome of treatment. 3, 14, 15

Dimensional changes after bone regeneration surgeries have been evaluated with CBCT technique. 3, 14, 15 Fiorellini et al.3 measured the alveolar ridge height and width changes on CBCT after extraction and the use of recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered on a bioabsorbable collagen sponge

(ACS) on test group compared to placebo in control group. In another study,14 ridge preservation with bone allograft was compared to no treatment after single non- molar tooth extraction. Radiographic stent with radiopaque markers was used as a reference on CBCT images to allow standardization of measurements of alveolar ridges. In both studies, all measurements were reported as linear measurements, and the differences were calculated between baseline and 3-4 months after treatment.

The purpose of this study was to evaluate ridge dimensional changes following socket preservation (SP) and Guided Bone Regeneration (GBR) surgeries using CBCT technique. Three different measurement protocols were used in order to determine ridge dimensional changes documented by comparing baseline and follow-up CBCTs. In addition, this study aimed to evaluate possible effects of wound healing and rate of blood perfusion on bone fill outcome.

75 Materials and Method:

Study design:

The study was a prospective observational clinical trial. Patients treatment planned for extraction and/or bone regeneration before implant placement for a single non-molar maxillary site were recruited. Clinical examination and sampling were conducted prior to surgery and at 3, 6, 9 days, 1 month and 4 months post-surgery. Clinical parameters, wound healing measures, GCF sample,

LDF readings and soft tissue biopsy were obtained at different time points. CBCT was obtained immediately after treatment and 4 months post-surgery. The

Institutional Review Board of The Ohio State University (OSU) approved the study protocol (protocol #2014H0150), data collection and informed consent forms.

Subject population:

Patient referred to the Graduate Periodontology Clinics at OSU for bone regeneration surgeries before implant placement in a single non-molar maxillary site were recruited. Inclusion criteria includes:

• Age between 18 and 65 years old

• Single non molar maxillary tooth site

• Stable periodontal condition

• Good systemic health

Exclusion criteria:

• Systemic condition that can affect wound healing

76 • Smoking

• Pregnancy

Signed informed consent was obtained from all subjects.

Surgical protocols, Clinical measures and Patient based outcomes:

Surgical protocols and methods related to clinical examination, wound fluid sampling, biopsies as well as techniques related to multiplex assays and, LDF readings were provided in Chapter III.

Clinical wound healing was evaluated by Visual analogue scale (VAS) as well as by using clinicalwound healing scores (0=Mature wound healing,

1=Erythema, 2=Bleeding, 3=Graft mobility, 4=Suppuration, 5=Necrosis)

(modified from Kloostra et al.16). Details related to these methods were provided in

Chapter III.

CBCT Scan:

The patients were scanned at baseline (1-3 days after the surgery) and at 4 months follow up. The same stent used for LDF readings (Chapter III) was used during CBCT scans. A radiopaque marker (Gutta Percha Point= GP) was added on the buccal side of the stent in the treatment site to be used as a reference point for measurements. CBCT images were obtained using i-CAT system (Imaging Sciences

International, Hatfield, PA). The CBCT unit was set at 8 × 8 cm FOV with 14.7 seconds exposure time. The generated images (DICOM-based data sets) had 0.2 mm voxel size. The DICOM data set were saved on hard disc and reconstructed using specific software (OsiriX Lite v.7.0.2 Pixmeo, Geneva, Switzerland.). This protocol

77 was established by an oral radiologist in order to reduce radiation while obtaining a detailed picture of the single tooth surgical area localized in maxillary anterior region. 3 different measurements, i.e. Linear (L), buccolingual width (BL) and 3- dimensional (3D) were performed on CBCTs obtained from the same patient immediately following surgery and at 4 months follow-up period. One examiner performed all measurements. To calculate possible CBCT image distortion, a stable structure was used and measured in the two CBCT image set. The measurements were compared using paired t-test.

Outcome measurements:

Linear measurements:

Linear measurement is used to measure the distance between a fixed point

(GP marker) and the buccal plate. Using the adjacent teeth as a reference, the middle of the edentulous area was identified. On the selected image section, a line (A) was drawn parallel to the buccal plate and through the most prominent part. Then a perpendicular line (B) dropped from the reference point to the line A (modified from Lee et al. 17). Line B represents the distance from the buccal plate, and can indicate bone dimensional changes on the buccal aspect of the ridge (Figure 21).

This measurement was done at baseline (L0) and repeated on the second data set taken at 4 months follow up (L1). The difference between the two measurements

(ΔL) was calculated as:

(ΔL) = L0-L1

This measurement was recorded in millimeters (mm).

78 Buccal-Lingual (BL) measurements:

BL measurement represents the bucco-lingual width of the ridge at the most coronal part of the ridge. This measurement used to evaluate whether there was adequate alveolar ridge bone width to support the placement of an endosseous dental implant at 4 months following treatment. Adequate alveolar bone was defined as ≥6 mm in width at the coronal part (buccal to palatal). In addition, the percentage of patients who required a secondary augmentation procedure was calculated.

Using the same section for the linear measurements, two lines were drawn parallel to the buccal and palatal plates (A & B). In addition, a third line (C) bisecting and parallel to the long access of the ridge was drawn. Finally, line (D) was drawn perpendicular on C and at the most coronal part between the two lines A & B. Line D represent the BL width of the ridge. (Modified from Fiorellini et al. 3) as seen in

Figure 22. This measurement was recorded in mm.

3-dimensional measurements:

The 3-D measurement represents the volume change in the grafted site. Due to difficulty to separate the grafted part from the native bone, the area measured included both teeth on each side of the treatment site as well as the total maxillary bone area visible in each image section. Although each measurement did not represent the actual width or height of the alveolus, subtraction of the 4 months measurement (D1) from the baseline measurement (D0) will give the dimensional change (ΔD) at the buccal, palatal and coronal aspect of the ridge.

79 Starting from the middle of the tooth on one side of the treatment site, the bone was manually traced on each section to the middle of the tooth on the other side. 40-60 sections were measured on each image data set rendering a 3-D object in each data set (Figure 23). Ridge dimensional change was calculated as follows:

ΔD= D1- D0

This measurement was recorded in cubic centimeters (cm3).

Data management and statistical analysis:

Clinical measurements were performed by a single trained examiner, using UNC-15 probe. Data was presented as mean ± standard error. LDF readings were calculated as an average of the 2 minutes period of each individual recording by the Periosoft computer program (PSW 2, version 2.5.5, Perimed AB).

Changes of blood flow values were expressed as the percent difference (ΔPU%).18,

19 CBCT measurements are presented as mean ± standard error. Linear and 3-D measurements were calculated as the difference between baseline and 4 months follow up.

The data were analyzed using both GraphPad Prism 5 (GraphPad Software,

Inc. CA, USA) and Statistical Analysis Software, version 9.3 (SAS Institute Inc., Cary,

NC, USA). Unpaired t-test or Mann-Whitney U test were used to compare CBCT data between SP and GBR group. Additionally, logistic regression analysis was performed to reveal the association between binary outcomes and other variables. Pearson correlation coefficients were calculated to reveal the association between various parameters. Differences were accepted as statistically significant at p≤0.05 level.

80 Results:

Demographics, clinical parameters, clinical wound healing, LDF data and wound fluid angiogenesis markers are presented in chapter III. On this section, we will focus on the CBCT data and its correlation with other variables.

CBCT data:

CBCT scans were taken from all subjects at baseline and 4 months follow up

(4.9 ± 1.1 months in SP and 4.5 ± 0.5 months in GBR). Total of 15 subjects completed the study with a total of 30 scans available for analysis. For one subject the CBCT scan was corrupted and could not be analyzed. Therefore only 28 scans for 14 subjects were included in the analysis. Figure 24 presents 2 cases, one from each group at baseline and 4 months after treatment. All measurements presented in tables 9 and 10. Comparison of the two CBCT data sets showed no significant difference between scans (P=0.29).

For linear measurements, the mean change (ΔL) in the SP group was - 0.75 ±

0.24 mm (ranging from -1.83 to 0.02 mm), while the ΔL in the GBR group was - 0.97

± 0.16 mm (ranging from – 1.33 to -0.47). The difference between the 2 groups was not statistically significant (p=0.50) (Figure 25).

For 3-D measurements, the mean change (ΔD) in SP group was – 0.22 ± 0.08 cm3 (ranging from – 0.69 to – 0.02 cm3). For GBR group, the ΔD was – 0.33 ± 0.07 cm3 (ranging from – 0.57 to – 0.11 cm3). The difference between the groups was statistically significant (p=0.05) (Figure 25).

For BL measurements, the mean ridge width at 4 months follow-up is 6.30 ±

81 0.40 mm and 6.50 ± 0.98 mm in SP and GBR groups, respectively (Table 10). The difference between the groups was negligeable (p=0.33). Based on CBCT scans, 50% of the patients in SP had enough bone width for implant placement (≥6 mm) at 4 months follow up compared to 67% in GBR group. At the end of the study period, 4 patients did not return for implant placement (1 subject has financial reasons and the other 3 patients had a change in their dental treatment planning). 11 patients (8 in SP group and 3 in GBR group) had second surgery at 4 months. 7 patients (5 in SP group and 2 in GBR group) needed additional grafting at 4 months follow up. In all cases grafting was done at the time of implant placement except for one patient (SP group), who needed another GBR procedure and no implant placement performed at 4 months follow up (Table 11).

Pearson correlation coefficients show no correlation between LDF readings

(ΔP%) and ΔL & ΔD measurements (r=0.16, 0.12 and -0.07, with p=0.11, 0.23 and

0.46 respectively) (Table 12).

Analysis of association of tissue biotype with CBCT measurements showed no correlation (P>0.05), as shown in Table 13. In addition, no correlation was found between CBCT measurements and primary wound closure at the end of the surgery

(P>0.05) (Table 14). However, linear regression analysis shows association between the amount of wound exposure mesio-distally (MD) and the difference in 3-D measurements (Table 15).

82 Discussion:

With increased popularity of dental implants, bone regeneration surgeries are widely used to improve ridge dimensions for implant placement. Outcomes of bone regeneration were reported clinically, radiographically and histologically. The aim of this study was to report dimensional ridge changes after SP and GBR surgeries using CBCT scans. We provided 3 different measurements to report the change or outcome from the baseline to 4 months follow up. Our main result wasbased on the volume change of the treatment site. Linear measurements were provided to better understand the fate of the buccal plate. Finally, the BL measurements were used to report the outcome in term of ability to place implant in the treatment site. Also the need for additional grafting at the site of treatment was reported as another important clinical outcome measure for bone regeneration surgeries.

3-dimensional volume measurement provides a better understanding of the changes in the treatment site. It reflects the changes on the whole area rather than a point or surface. However, it does not identify the location of the bone remodeling.

Since we were interested on the buccal plate, we included a second measurement

(linear) that can provide an insight to the area of change. Linear measurements only reflect the change that occurs at a specific point on a selected section. Therefore, does not provide an accurate estimate of the change in the area. For these reasons, linear measurement was included only as supplementary information in addition to

3-D measurements. It is also important to report clinically relevant information in

83 terms of bone regeneration outcomes. BL measurements provide important information regarding the ability to place implant on the area. It is easy to interpret and apply in clinical setting.

In the 3-D method, the area measured was not only limited to the bone graft area but also included the surrounding bone and teeth due to difficulty in differentiation between bone graft and native bone. This still allowed us to measure changes in the treatment site. However, the measured change can be underestimated by increasing the volume of the area. Another possible solution will be to use density measurements to better visualize bone graft material and separate it from native bone.

In our study, we report ridge dimensional change after SP as well as GBR surgeries. Our finding is in agreement with other studies, which reported that bone regeneration could not prevent bone resorption after tooth extraction.2, 6-9 3-D measurements showed mean volume change of -0.22 cm3 (8.5% reduction) in SP group and – 0.33 cm3 (11.3 % reduction) in GBR group. Volumetric changes after bone grafting surgeries have been reported in few other studies. 15, 20, 21 All of these studies used block autogenous or allogenic grafts. Smolka21 reported 16.2% volume reduction 6 months after alveolar ridge reconstruction with calvarial split bone grafts. When iliac crest or chin bone grafts were used, the mean volume resorption after 1 year was 35-50%.20 The difference between the reported change in our study and the other studies can be due to the use of different graft material. Most of the studies report the use autogenous block graft that undergoes extensive resorption

84 during healing.22, 23 Healing time can be another significant factor that contributes to the discrepancy between studies. Our results are reported after an average of 4.7 ±

0.9 months of healing, while in other studies evaluation was done at 6 months or 1 year after treatment. Finally, in our study we only included single tooth area for treatment, while others reported mostly treatment of extensively resorbed alveolar ridges.

Volume change in GBR group was statistically more significant compared to

SP group. This can be attributed to the difference in case selection and surgical approach between the 2 groups. Sites were treated with SP if the socket walls were intact, which allows placement of bone graft materials without the need for barrier membrane. On the other hand, when bone loss was more extensive in the treatment site with loss of socket walls, GBR was the treatment of choice. In addition, GBR surgeries involved full thickness flap elevation with or without vertical incisions resulting in more surgical trauma compared to SP group. Although a limited flap elevation performed in SP group to make the two groups comparable, the flap was limited on the mid buccal of the extraction socket.

Linear measurements were used to calculate the distance between a reference point and the buccal aspect of the ridge. Therefore, it is a measure of bone change relative to buccal plate. Our results showed some dimensional change which indicates bone resorption on the buccal site. This is in agreement with other studies that report that most of the changes after extraction occur at the expense of the buccal plate.24, 25

85 The outcome of the bone regeneration surgery was not affected by the blood perfusion of the overlying flap. Although LDF readings can reflect the gingival blood perfusion, they cannot measure the vascularity of the undelying bone. Fullerton et al.26 showed that LDF light penetrates the tissue variably, to a depth of 0.6 mm. therefore, LDF reflects the blood perfusion at the supra-periosteal gingival plexus only. This can explain in part the lack of correlation between LDF readings and bone fill outcome.

Tissue biotype did not seem to influence the amount of bone fill after bone regeneration surgeries. Although some studies27, 28 showed positive association between gingival thickness and bone thickness measured radiographically, tissue biotypes have been reported as a significant factor in soft tissue healing. Initial gingival thickness was found to be the most significant factor associated with a complete root-coverage following soft tissue augmentation procedure.29 In immediate single-tooth implant restorations, patients with thin biotype often had more tissue recession,30 while those with thick biotype tended to maintain the implant papillae height. 31

According to our findings, wound closure did affect the amount of bone fill after regenerative procedure. This is in agreement with earlier reports32, 33 that showed compromised bone regeneration with early exposure of the site during healing period. However, more recent studies34-36 documented uneventful healing and bone regeneration in sites that did not have primary closure at the time of surgery.

86 This study has some limitations. First, the sample number is small. Second, the CBCT setting was modified to reduce the radiographic exposure to acceptable limits for the purposes of this study. This modification reduced the quality of the obtained images and made the tracing occasionally difficult. However, changing image contrast improved the visualization and made it readable. Finally, the area measured in the 3-D method was not limited to the bone graft area, but extended to the adjacent teeth and included the whole maxillary bone visible in each section.

This apparently can underestimate the amount of change reported relative to the area measured.

In conclusion, within the limits of this study, alveolar ridges undergo bone resorption after SP and GBR surgeries. The amount of bone remodeling is more significant in GBR cases. A trend observed for a change in the expense of buccal plate. Bone regeneration outcome is not affected by the gingival blood perfusion levels or tissue biotype. Wound closure during early healing period may affect the bone fill outcomes.

87

A

B

Figure 21: Linear measurement; line (A) is drawn parallel to the buccal plate and line (B) dropped from the reference point perpendicular to the line A. line B presents the distance from the buccal plate.

A C

D B

Figure 22: BL measurement; two lines were drawn parallel to the buccal and palatal plates (A & B). In addition, a third line (C) bisecting and parallel to the long access of the ridge was drawn. Line (D) was drawn perpendicular on C and at the most coronal part between the two lines A & B. Line D represent the BL width of the ridge.

88

Figure 23: 3-D object.

Figure 24. GBR case (top) at baseline (left) and 4 months follow up (right) and SP

case (bottom) at baseline (left) and 4 months follow up (right) 89

SP (n=8) GBR (n=6) p-value

ΔL (mm) - 0.75 ± 0.24 - 0.97 ± 0.16 0.50

ΔD (cm3) – 0.22 ± 0.08 – 0.33 ± 0.07 0.05 *

Table9 : differences between baseline and 4 months follow up in linear and 3- dimensional measurements. ΔL: difference in linear measurements, ΔD: difference in 3-D measurements

SP (n=8) GBR (n=6) p-value

BL (mm) 6.30 ± 0.40 6.50 ± 0.98 0.33 Table 10: width of the ridge at 4 months post-operative visit. BL: bucco-lingual

*

Figure 25: comparing SP and GBR in the change in linear and 3-dimensional measurements between baseline and 4 months. ΔL: difference in linear measurements (mm) and ΔD: difference in 3-D measurements (cm3), ΔD is significant between the two groups * P=0.05

90

SP (n=8) GBR (n=6)

No implant placement 1* 3

Additional augmentation 4 2

(At the time of implant placement)

No augmentation 3 1

Table 11: outcome of bone regeneration surgeries. 4 patients (3 GBR & 1SP) did not schedule implant placement surgery yet * Patient had a second bone augmentation procedure

Bone changes r p-value

ΔL 0.16 p 0.11 ΔP% ΔD 0.12 0.23

Table 12: correlation between LDF data and bone changes. ΔL: difference in linear measurements, ΔD: difference in 3-D measurements, BL: bucco-lingual width between baseline and 4 months

CBCT Tissue biotype P-value

Thin Thick

ΔL -0.98 ± 0.65 -0.70 ± 0.45 0.383

ΔD -0.18 ± 0.18 -0.12 ± 0.14 0.477

Table 13: correlation between tissue biotype and bone changes at 4 months ΔL: difference in linear measurements, ΔD: difference in 3-D measurements.

91

CBCT Primary closure p-value No Yes

ΔL -0.87 ± 0.60 -0.74 ± 0.43 0.733

ΔD -0.13 ± 0.15 -0.22 ± 0.18 0.415

Table 14: correlation primary wound closure and bone changes at 4 months. ΔL: difference in linear measurements, ΔD: difference in 3-D measurements.

CBCT Wound Closure Estimate p-value

ΔL Exposure MD -0.011 0.920

ΔL Exposure BL -0.050 0.639

ΔD Exposure MD -0.064 0.046*

ΔD Exposure BL -0.055 0.074

Table 15: correlation between wound closure and bone changes at 4 months. ΔL: difference in linear measurements, ΔD: difference in 3-D measurements, MD: mesio- distal exposure, BL: bucco-lingual exposure.

92

References:

1. Aimetti M, Romano F, Griga FB, Godio L. Clinical and histologic healing of human extraction sockets filled with calcium sulfate. Int J Oral Maxillofac Implants 2009;24:902-909. 2. Barone A, Aldini NN, Fini M, Giardino R, Calvo Guirado JL, Covani U. Xenograft versus extraction alone for ridge preservation after tooth removal: a clinical and histomorphometric study. Journal of periodontology 2008;79:1370-1377. 3. Fiorellini JP, Howell TH, Cochran D, et al. Randomized study evaluating recombinant human bone morphogenetic protein-2 for extraction socket augmentation. Journal of periodontology 2005;76:605-613. 4. Froum S, Cho SC, Rosenberg E, Rohrer M, Tarnow D. Histological comparison of healing extraction sockets implanted with bioactive glass or demineralized freeze-dried bone allograft: a pilot study. Journal of periodontology 2002;73:94-102. 5. Hoad-Reddick G, Grant AA, McCord JF. Osseoretention? Comparative assessment of particulate hydroxyapatite inserted beneath immediate dentures. Eur J Prosthodont Restor Dent 1994;3:61-65. 6. Iasella JM, Greenwell H, Miller RL, et al. Ridge preservation with freeze-dried bone allograft and a collagen membrane compared to extraction alone for implant site development: a clinical and histologic study in humans. Journal of periodontology 2003;74:990-999. 7. Lekovic V, Camargo PM, Klokkevold PR, et al. Preservation of alveolar bone in extraction sockets using bioabsorbable membranes. Journal of periodontology 1998;69:1044-1049. 8. Lekovic V, Kenney EB, Weinlaender M, et al. A bone regenerative approach to alveolar ridge maintenance following tooth extraction. Report of 10 cases. Journal of periodontology 1997;68:563-570. 9. Vignoletti F, Matesanz P, Rodrigo D, Figuero E, Martin C, Sanz M. Surgical protocols for ridge preservation after tooth extraction. A systematic review. Clin Oral Implants Res 2012;23 Suppl 5:22-38. 10. De Vos W, Casselman J, Swennen GR. Cone-beam computerized tomography (CBCT) imaging of the oral and maxillofacial region: a systematic review of the literature. Int J Oral Maxillofac Surg 2009;38:609-625. 11. Mah JK, Danforth RA, Bumann A, Hatcher D. Radiation absorbed in maxillofacial imaging with a new dental computed tomography device. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;96:508-513. 12. Kasaj A, Willershausen B. Digital volume tomography for diagnostics in periodontology. Int J Comput Dent 2007;10:155-168. 13. Orentlicher G, Goldsmith D, Abboud M. Computer-guided planning and placement of dental implants. Atlas Oral Maxillofac Surg Clin North Am 2012;20:53-79. 93 14. Brownfield LA, Weltman RL. Ridge preservation with or without an osteoinductive allograft: a clinical, radiographic, micro-computed tomography, and histologic study evaluating dimensional changes and new bone formation of the alveolar ridge. Journal of periodontology 2012;83:581- 589. 15. Spin-Neto R, Stavropoulos A, Dias Pereira LA, Marcantonio E, Jr., Wenzel A. Fate of autologous and fresh-frozen allogeneic block bone grafts used for ridge augmentation. A CBCT-based analysis. Clin Oral Implants Res 2013;24:167-173. 16. Kloostra PW, Eber RM, Wang HL, Inglehart MR. Surgical versus non-surgical periodontal treatment: psychosocial factors and treatment outcomes. Journal of periodontology 2006;77:1253-1260. 17. Lee M, Kanavakis G, Miner RM. Newly defined landmarks for a three- dimensionally based cephalometric analysis: a retrospective cone-beam computed tomography scan review. Angle Orthod 2015;85:3-10. 18. Donos N, D'Aiuto F, Retzepi M, Tonetti M. Evaluation of gingival blood flow by the use of laser Doppler flowmetry following periodontal surgery. A pilot study. Journal of periodontal research 2005;40:129-137. 19. Zanetta-Barbosa D, Klinge B, Svensson H. Laser Doppler flowmetry of blood perfusion in mucoperiosteal flaps covering membranes in bone augmentation and implant procedures. A pilot study in dogs. Clin Oral Implants Res 1993;4:35-38. 20. Sbordone L, Toti P, Menchini-Fabris GB, Sbordone C, Piombino P, Guidetti F. Volume changes of autogenous bone grafts after alveolar ridge augmentation of atrophic maxillae and mandibles. Int J Oral Maxillofac Surg 2009;38:1059- 1065. 21. Smolka W, Eggensperger N, Carollo V, Ozdoba C, Iizuka T. Changes in the volume and density of calvarial split bone grafts after alveolar ridge augmentation. Clin Oral Implants Res 2006;17:149-155. 22. Johansson B, Grepe A, Wannfors K, Hirsch JM. A clinical study of changes in the volume of bone grafts in the atrophic maxilla. Dentomaxillofac Radiol 2001;30:157-161. 23. Nystrom E, Legrell PE, Forssell A, Kahnberg KE. Combined use of bone grafts and implants in the severely resorbed maxilla. Postoperative evaluation by computed tomography. Int J Oral Maxillofac Surg 1995;24:20-25. 24. Araujo MG, Lindhe J. Dimensional ridge alterations following tooth extraction. An experimental study in the dog. Journal of clinical periodontology 2005;32:212-218. 25. Pietrokovski J, Massler M. Alveolar ridge resorption following tooth extraction. J Prosthet Dent 1967;17:21-27. 26. Fullerton A, Stucker M, Wilhelm KP, et al. Guidelines for visualization of cutaneous blood flow by laser Doppler perfusion imaging. A report from the Standardization Group of the European Society of Contact Dermatitis based

94 upon the HIRELADO European community project. Contact Dermatitis 2002;46:129-140. 27. Fu JH, Yeh CY, Chan HL, Tatarakis N, Leong DJ, Wang HL. Tissue biotype and its relation to the underlying bone morphology. Journal of periodontology 2010;81:569-574. 28. Stein JM, Lintel-Hoping N, Hammacher C, Kasaj A, Tamm M, Hanisch O. The gingival biotype: measurement of soft and hard tissue dimensions - a radiographic morphometric study. Journal of clinical periodontology 2013;40:1132-1139. 29. Baldi C, Pini-Prato G, Pagliaro U, et al. Coronally advanced flap procedure for root coverage. Is flap thickness a relevant predictor to achieve root coverage? A 19-case series. Journal of periodontology 1999;70:1077-1084. 30. Evans CD, Chen ST. Esthetic outcomes of immediate implant placements. Clin Oral Implants Res 2008;19:73-80. 31. Romeo E, Lops D, Rossi A, Storelli S, Rozza R, Chiapasco M. Surgical and prosthetic management of interproximal region with single-implant restorations: 1-year prospective study. Journal of periodontology 2008;79:1048-1055. 32. Machtei EE. The effect of membrane exposure on the outcome of regenerative procedures in humans: a meta-analysis. Journal of periodontology 2001;72:512-516. 33. Simion M, Misitano U, Gionso L, Salvato A. Treatment of dehiscences and fenestrations around dental implants using resorbable and nonresorbable membranes associated with bone autografts: a comparative clinical study. Int J Oral Maxillofac Implants 1997;12:159-167. 34. Barone A, Ricci M, Tonelli P, Santini S, Covani U. Tissue changes of extraction sockets in humans: a comparison of spontaneous healing vs. ridge preservation with secondary soft tissue healing. Clin Oral Implants Res 2013;24:1231-1237. 35. Beck TM, Mealey BL. Histologic analysis of healing after tooth extraction with ridge preservation using mineralized human bone allograft. Journal of periodontology 2010;81:1765-1772. 36. Cardaropoli D, Cardaropoli G. Preservation of the postextraction alveolar ridge: a clinical and histologic study. Int J Periodontics Restorative Dent 2008;28:469-477.

95

Chapter 5

Conclusion

The results of our study demonstrated that gingival blood perfusion presents an ischemic-reperfusion model. A hyperemic response was seen during early healing period followed by a decrease in blood perfusion levels, however, it sustained higher levels compared to baseline after bone regeneration surgeries. In addition, although there is no statistical significant difference between the two groups, blood perfusion showed more fluctuation and slower recovery in GBR group compared to SP group. There is statistically significant difference between the two groups in IL-8 and TGF-α levels during early healing period, however, SP group showed delayed expression of angiogenic markers. No correlation between angiogenic markers and blood perfusion level exists. Blood perfusion is affected by the wound closure. Soft tissue biotype doesn’t have statistically significant effect on blood perfusion levels. On hard tissue level, alveolar ridges undergo bone resorption after both SP and GBR surgeries. The amount of bone remodeling is more significant in GBR cases. Dimensional bone change is seen on the buccal aspect of the ridge. Bone regeneration outcomes is not affected by the gingival blood perfusion levels or tissue biotype, however, wound closure during early healing period may affect the bone fill outcomes.

96

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101 • De Vos W, Casselman J, Swennen GR. Cone-beam computerized tomography (CBCT) imaging of the oral and maxillofacial region: a systematic review of the literature. Int J Oral Maxillofac Surg 2009;38:609-625. • Mah JK, Danforth RA, Bumann A, Hatcher D. Radiation absorbed in maxillofacial imaging with a new dental computed tomography device. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;96:508-513. • Kasaj A, Willershausen B. Digital volume tomography for diagnostics in periodontology. Int J Comput Dent 2007;10:155-168. • Orentlicher G, Goldsmith D, Abboud M. Computer-guided planning and placement of dental implants. Atlas Oral Maxillofac Surg Clin North Am 2012;20:53-79. • Brownfield LA, Weltman RL. Ridge preservation with or without an osteoinductive allograft: a clinical, radiographic, micro-computed tomography, and histologic study evaluating dimensional changes and new bone formation of the alveolar ridge. Journal of periodontology 2012;83:581- 589. • Spin-Neto R, Stavropoulos A, Dias Pereira LA, Marcantonio E, Jr., Wenzel A. Fate of autologous and fresh-frozen allogeneic block bone grafts used for ridge augmentation. A CBCT-based analysis. Clin Oral Implants Res 2013;24:167-173. • Kloostra PW, Eber RM, Wang HL, Inglehart MR. Surgical versus non-surgical periodontal treatment: psychosocial factors and treatment outcomes. Journal of periodontology 2006;77:1253-1260. • Lee M, Kanavakis G, Miner RM. Newly defined landmarks for a three- dimensionally based cephalometric analysis: a retrospective cone-beam computed tomography scan review. Angle Orthod 2015;85:3-10. • Sbordone L, Toti P, Menchini-Fabris GB, Sbordone C, Piombino P, Guidetti F. Volume changes of autogenous bone grafts after alveolar ridge augmentation of atrophic maxillae and mandibles. Int J Oral Maxillofac Surg 2009;38:1059- 1065. • Smolka W, Eggensperger N, Carollo V, Ozdoba C, Iizuka T. Changes in the volume and density of calvarial split bone grafts after alveolar ridge augmentation. Clin Oral Implants Res 2006;17:149-155. • Johansson B, Grepe A, Wannfors K, Hirsch JM. A clinical study of changes in the volume of bone grafts in the atrophic maxilla. Dentomaxillofac Radiol 2001;30:157-161. • Nystrom E, Legrell PE, Forssell A, Kahnberg KE. Combined use of bone grafts and implants in the severely resorbed maxilla. Postoperative evaluation by computed tomography. Int J Oral Maxillofac Surg 1995;24:20-25.

102