Blood Perfusion and Early Wound Healing Following Implant Placement

Blood Perfusion and Early Wound Healing Following Implant Placement:

A Comparison Between Grafted and Non-Grafted Sites

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of

Science in the Graduate School of The Ohio State University

Vrisiis Kofina, DDS

Graduate Program in Dentistry

The Ohio State University

2018

Thesis Committee:

Dr. Binnaz Leblebicioglu, Advisor

Dr. Dimitris Tatakis

Dr. Barbaros Selnur Erdal Copyright by

Vrisiis Kofina, DDS

2018

Abstract

Objective: This study aimed to determine the rate of recovery from surgical trauma through blood perfusion following implant placement surgery and to develop a reliable method to determine volumetric and linear changes within buccal bone following implant placement surgeries using Cone Beam Computed Tomography

(CBCT). CBCT-related results were compared with previously used technique to evaluate regenerated bone volume within similar anatomical location.

Materials and methods: Patients receiving single non-molar implant in the maxillary esthetic zone were recruited. Data collection was performed at the day of surgery; 3, 6, 9 days; 1 and 4 months, postoperatively. Soft tissue healing was recorded using well-established wound healing parameters during the entire healing period. Wound fluid collected at 3, 6 and 9 days and gingival crevicular fluid

(GCF) collected at baseline, 1 and 4 months from the adjacent teeth was used for future multiplex analysis of metabolism mediators. Soft tissue biopsies were collected from the wound site at baseline, 9 days and 4 months and stored for future bone remodeling related gene expression analysis. Buccal flap blood perfusion using

Laser Doppler Flowmetry (LDF) was recorded immediately before and after implant placement surgery, at 3, 6, 9 days and, 1 and 4 months. Soft tissue biotype and

ii implant stability quotient (ISQ) were determined at the time of implant placement and at 4 months. Bone thickness changes along the peri-implant buccal wall were evaluated by automatic superimposition of two CBCTs taken immediately after surgery and at 4 months. Peri-implant buccal bone changes along the length of the implant were calculated by using a software. Buccal bone grey values were selected based on soft tissue and bone grey values after automatic superimposition of images. Patients receiving extraction and bone regeneration [socket preservation

(SP) or guided bone regeneration (GBR)] within similar anatomical location were recruited. Two CBCTs were taken with a timeline as described above. Buccolingual, linear and volumetric measurements targeting buccal bone thickness were performed manually by using standard marks embedded into surgical guides or by using adjacent teeth to define the area of interest while comparing two CBCTs obtained from the same subject. The advantages and disadvantages of both techniques were discussed. Descriptive statistics are reported as mean±se and percentage. Patient was chosen as unit of measurement. A linear mixed model regression analysis was used for repeated continuous measures fixed and random effects within and between groups. A random effect (intercept and slope) regression analysis was conducted to estimate the slopes of the outcome over continuous time for non-grafted and grafted groups. For repeated measure binary outcomes, generalized estimating equations (GEEs) was used. T-test, chi-square test or Wilcoxon-Man-Whitney test, as appropriate, was used to analyze all the other

iii non-repeated data. Spearman’s correlation coefficient was used for correlation analyses.

Results: 24 patients (49±4 years; 12 males; single implant site; 9 non-grafted cases) completed the study. Clinical healing was uneventful in all cases. Soft tissue closure over the implant was slower in grafted group (p<0.01 between groups). Wound fluid volume increase was more persistent in grafted sites, peaking at 3 (p<0.01) and 6 days (p=0.04), compared to non-grafted sites (peak at 6 days; <0.01). Both groups reached baseline GCF levels by 9 days. In grafted group only, thin tissue biotype was correlated with higher wound fluid production (r=0.4, p=0.03). Blood perfusion decreased significantly immediately postoperatively in both groups

(p<0.01 within both groups). At 3 days the blood perfusion recovery was 57% and

42% in the non grafted and the grafted group, respectively. Although non-grafted sites had a 4-month recovery level comparable to 3 days post-op (p>0.05), grafted sites remained relatively ischemic at 4 months (p<0.01). 56% of sites were initially diagnosed as thin biotype. Mean ISQ increased from 69±4 and 69±1 to 72±3 and

74±2 in non-grafted and grafted sites, respectively (p<0.01 in grafted group only).

Initial buccal bone thickness for non-grafted and grafted sites was 1.52±0.05 mm

(1.3-1.6 mm) and 1.78±0.1 mm (1.5-2.1 mm), respectively. At 4 months, mean loss of 0.09±0.008 mm (0.06-0.1 mm) and 0.3±0.06 mm (0.1-0.5 mm) was evident in non-grafted and grafted sites, respectively (p<0.01, between groups). Thick soft tissue biotype was correlated with less buccal bone thickness loss at the apical 10-

iv 12mm along the implant length in both groups (r=0.8, p=0.03). 13 CBCT records following post-tooth extraction and bone regeneration procedures were collected.

Linear distance measurements from the surgical stent to ridge revealed - 0.75 ± 0.24 mm buccal bone loss in SP and - 0.97 ± 0.16 mm buccal bone loss in GBR group, respectively. Mean 3-D bone volume changes, determined by using adjacent teeth as standardization points and subtracting buccal half of the ridge for calculations, revealed volumetric bone loss of – 0.22 ± 0.08 cm3 and – 0.33 ± 0.07 cm3 in SP and

GBR groups, respectively (p=0.05 between groups).

Conclusion: Post-implant placement early wound healing in grafted sites is characterized by a continuous ischemic response and a higher inflammatory clinical healing profile compared to less surgically manipulated non-grafted sites. However, despite surgical trauma, buccal bone thickness loss is minimal at both grafted and non-grafted sites and consistent with previously reported bone thickness loss after flap elevation. Computerized volumetric evaluation and automatic selection of the areas of interest following CBCT image superimposition allow for better evaluation of bone changes compared to manual area selection as long as radiation exposure and dental implant related artifacts can be controlled.

Dedication

Dedicated to my Family, Alex and my Teachers

vi Acknowledgments

I would like to express my gratitude to Dr. Binnaz Leblebicioglu for her passion, guidance and care while introducing me to clinical research and teaching me how to develop research protocols and analyze data. I would like to thank Dr. Dimitris

Tatakis for his devotion to this project, help with ideas for data analysis and support with abstract corrections and competitions. I would also like to acknowledge Dr.

Barbaros Selnur Erdal for his help with the CBCT part of the study. I would like to express my sincere appreciation to Dr. Mutlu Demirer for his tireless effort, ideas and time with the CBCT analysis of this project. I wish to thank Dr. Eubank for his generous assistance with the cytokine analysis in his lab and Dr. Brian L. Foster for his insightful guidance with biopsy analysis and for providing his lab facility space and equipment. I would also like to thank Dr. Lamees Alssum for training me for the clinical measurements of the study, Michael Chaves and Michelle Tan for helping me with lab work and Debbi Pack for taking the CBCTs in the Radiology Clinic.

Furthermore, I would like to thank all the Graduate Periodontal Residents for their support with recruitment. The study was supported by intramural (OSU COD) and extramural (AAIDF) grants to Dr. Binnaz Leblebicioglu.

vii Vita

2013…………… Doctor of Dental Surgery, National & Kapodistrian University of

Athens, Greece

2014…………………………………………………………………………Private Practice Employment

2015-2018…………………… Post-doctoral Training in Periodontics, The Ohio State

University, The Ohio State University, Columbus, Ohio, USA

Peer-reviewed abstracts

§ CBCT analysis of post-surgery buccal bone thickness

Leblebicioglu B., Kofina V., Demirer M., Alssum L., Erdal B.S., Yildiz V.O.,

Tatakis D.N.

Poster accepted at EuroPerio9, Amsterdam, The Netherlands – 06/20/2018 -

06/23/2018

§ Bone grafting and post-implant surgery flap blood perfusion rate

Kofina V., Demirer M., Alssum L., Erdal B.S., Yildiz V.O., Tatakis D.N.,

Leblebicioglu B.

Poster at 2018 AADR/CADR, Fort Lauderdale, FL, USA – 03/24/2018

§ Buccal bone thickness in relation to flap blood perfusion and implant

stability

viii Kofina V., Demirer M., Alssum L., Erdal B.S., Yildiz V.O., Tatakis D.N.,

Leblebicioglu B.

Oral presentation at 2018 Midwest Society of Periodontology Graduate

Student Research Forum (1st Honorary Mention), Chicago, IL, USA –

2/24/2018

§ CBCT analysis of alveolar bone remodeling following regenerative surgery

Kofina V., Alssum L., Erdal B.S., Yildiz V.O., Tatakis D.N., Leblebicioglu B.

Poster at Research Day of COD at OSU, 10th Annual Translational to Clinical

Regenerative Medicine Wound Care Conference, Columbus, OH, USA –

02/28/2017 & 03/10/2017 and 2017 IADR/AADR/CADR, San Francisco, CA,

USA – 03/24/2017

§ Potential risk predictors for peri-implant diseases: An observational

prospective study

Heming Z., Kofina V., Sakulpaptong W., Yildiz V.O., Tatakis D.N.,

Leblebicioglu B.

Poster at Research Day of COD at OSU, Columbus, OH, USA – 02/28/2017 &

2017 IADR/AADR/CADR, San Francisco, CA, USA – 03/24/2017

§ Modified periodontal indices and peri-implant diagnosis

Barriere T., Sakulpaptong W., Kofina V., Yildiz V.O., Tatakis D.N.,

Leblebicioglu B.

Poster at Research Day of COD at OSU, Columbus, OH, USA – 02/28/2017 &

2017 IADR/AADR/CADR, San Francisco, CA, USA – 03/24/2017

ix Table of contents

Abstract ...... ii

Dedication ...... vi

Acknowledgments ...... vii

Vita ...... viii

Table of Contents ...... x

List of Tables ...... xii

List of Figures ...... xiii

Chapter I: Introduction ...... 1

• Introduction ...... 1

• References ...... 11

Chapter II: Maxillary Buccal Bone Dimensional Measurement by CBCT:

Development of a method to determine dimensional changes following dental

implant placement surgery ...... 15

• Abstract ...... 15

• Introduction ...... 18

• Materials and Methods ...... 20

• Statistical Analysis ...... 27

• Results ...... 28

x • Discussion ...... 29

• Conclusion ...... 33

• References ...... 41

Chapter III: Blood perfusion and early healing following implant placement - A

comparison between grafted and non grafted sites ...... 43

• Abstract ...... 43

• Introduction ...... 47

• Materials and Methods ...... 50

• Statistical Analysis ...... 57

• Results ...... 58

• Discussion ...... 66

• Conclusion ...... 73

• References ...... 83

Chapter IV: conclusion ...... 86

• Conclusion ...... 86

• References ...... 90

xi List of tables

Table 1: Demographics for grafted and non-grafted groups ...... 75

xii List of Figures

Figure 1: Buccolingual measurement ...... 34

Figure 2: Linear measurement ...... 35

Figure 3: 3-D Object ...... 35

Figure 4: GBR case (top) at baseline (left) and 4 months (right) and SP case (bottom)

at baseline (left) and 4 months (right) ...... 36

Figure 5: Minimization of mean squared error for image registration of

subject #4 ...... 36

Figure 6: CBCT images of subject #8 immediately (blue) and 4 months (red) after

implant placement #11 ...... 37

Figure 7: Region of interest (ROI) ...... 37

Figure 8: Magnification of selected ROI from figure 7 – Implant Region of Interest

(Implant ROI) ...... 38

Figure 9: Magnification of image C of figure 8 – Buccal bone selection ...... 38

Figure 10: Soft tissue control area ...... 39

Figure 11: Bone control area ...... 39

Figure 12: Initial peri-implant buccal bone thickness heat map of subject #19 ...... 40

Figure 13: Peri-implant buccal bone thickness difference between first and second

CBCT (CBCT2-CBCT1) heat map of subject #19 ...... 40

xiii Figure 14: Plaque and gingival bleeding in treatment sextant ...... 75

Figure 15: Wound healing parameters in non-grafted sites (A) and grafted

sites (B) ...... 76

Figure 16A: VAS – Pain outcomes in non-grafted sites (A) and grafted sites (B) ...... 77

Figure 17: GCF (outlined in purple) and wound fluid (outlined in green) volume..... 78

Figure 18: LDF data for the total sample (N=25) ...... 78

Figure 19: LDF data for non-grafted (N=9) and grafted group (N=16) ...... 79

Figure 20: LDF data of non-grafted (N=9) and grafted group (N=16) of current study

and post-extraction bone regeneration group (N=15) from Alssum et al 41 ...... 79

Figure 21: Implant stability in non-grafted and grafted group ...... 80

Figure 22: Peri-implant buccal bone thickness for the total sample (N=24) at

baseline and at 4 months ...... 80

Figure 23: Per-implant buccal bone thickness for the total sample (N=24) along the

implant length at baseline and at 4 months ...... 81

Figure 24: Peri-implant buccal bone thickness in non-grafted (A; N=9) and grafted

group (B; N=15) at baseline and 4 months ...... 81

Figure 25: Peri-implant buccal bone loss in non-grafted (N=9) and grafted group

(N=15) between baseline and 4 months ...... 82

Figure 26: Peri-implant buccal bone loss in non-grafted (N=9) and grafted group

(N=15) ...... 82

xiv Chapter I

Introduction

The alveolar bone ridge undergoes changes throughout life with the most significant changes occurring after tooth extraction.1 Immediately after extraction the socket is filled with blood allowing for blood clot formation. During the first week, the blood clot is gradually replaced by granulation tissue starting from the periphery of the socket. After 2-4 weeks, provisional matrix replaces residues of severed periodontal ligament fibers, blood clot and granulation tissue and an epithelialized, well-organized connective tissue seal is formed to cover the coronal entrance of the socket. Within 6-8 weeks, the majority of the granulation tissue has been resorbed and replaced by provisional matrix. In the meantime, bone remodeling occurs with resorption of bundle bone and formation of woven bone.2

Bone resorption initially occurs at the crestal bone level especially on the buccal aspect of the socket and later on, along the outer surfaces of the buccal and lingual walls of the socket.3 After 12-24 weeks of healing, provisional matrix and woven bone mainly occupy the socket, with little lamellar bone formation. In human healing socket biopsies, lamellar bone does not usually appear in the socket before

12-24 weeks of healing.2

1 Clinically these changes correspond to alveolar bone dimension loss after tooth extraction. According to a systematic review by Tan et al4, 6 months after extraction the magnitude of vertical and horizontal alveolar bone loss reaches 11-

22% and 29-63% respectively with the midbuccal portion of the socket losing more bone than the other sites.5 The majority of the reduction occurs during the first 3-6 months and is followed by slower dimension changes thereafter. 4

The wide range of alveolar bone dimension changes is representative of the variety of factors affecting bone remodeling after extraction. In a cross sectional study, Leblebicioglu et al6 underlined the significance of the mechanical stress during “atraumatic” extractions as expressed by the alveolar ridge alterations (ridge width expansion and buccal bone integrity loss) in 2/3 of the sites immediately after tooth extraction. Multi-rooted teeth tended to show more alveolar ridge loss; however root morphology does not seem to be a determinant of vertical bone loss.7

In a prospective study by Chappuis et al8, initial buccal bone thickness was identified as a determinant of vertical alveolar bone loss in the esthetic zone. Initial buccal bone wall thickness of ≤ 1mm was associated with a median horizontal and vertical bone loss of 0.8mm and 7.5mm respectively, whereas buccal bone walls that were > 1mm thick exhibited a median horizontal and vertical bone loss of 0mm and

1.1mm respectively.8 This finding along with the fact that > 75% of the buccal bone walls are ≤ 1mm thick in the maxillary anterior and premolar teeth and >70% of the buccal bone walls are ≤ 0.5mm thick in anterior teeth, indicates the higher risk for significant alveolar bone dimension changes in the anterior region.9

2 In order to minimize the extent of bone loss after tooth extraction, different surgical protocols have been used such as flapless “atraumatic” tooth extraction and alveolar ridge preservation.7,10, 11 Even though there is no clear definition and the term is self-evident, “atraumatic” tooth extraction aims at minimizing the mechanical stress that is exerted on the bone while using elevators and forceps. This term, however, is controversial because the extraction itself is an invasive procedure. In order to evaluate the effect of flap elevation during “atraumatic extraction”, Fickl et al. performed flapless extractions and extractions after elevation of a full thickness flap for 10 minutes in dogs and concluded that extraction after flap elevation is associated with significantly more bone loss compared to flapless extraction.11

Alveolar ridge preservation refers to the procedure of placing materials in the socket after extraction in order to minimize the amount of bone loss.12 In cases when all socket walls are intact, bone graft material is placed in the socket acting as a space maintenance device. This procedure is called socket preservation.12 In cases of severe socket bone wall deficiency, the placement of a membrane over the bone graft material (guided bone regeneration) is indicated.13 Guided bone regeneration is based on the principle of selectively allowing osteogenic cells to differentiate and proliferate into the wound area promoting new bone formation.14 However, alveolar bone loss is not prevented after extraction and socket preservation or guided bone regeneration. In a systematic review by Ten Heggeler,15 3 months after extraction and socket preservation sites exhibited at least 1.12mm and 1.26mm less alveolar

3 bone width and height reduction respectively compared to sites that had extraction alone. In a systematic review by Horvath,16 up to 9 months after extraction and guided bone regeneration, the alveolar ridge width reduction ranged from 1.2-

2.3mm compared to 2.6-4.6mm in sites after extraction alone. The alveolar bone height changes ranged between an augmentation of 1.3mm to a reduction of 0.7mm after extraction and guided bone regeneration compared to a reduction of 0.9-

3.6mm after extraction alone.16

Implant placement surgery generally takes place 4-6 months following tooth extraction with/without hard tissue augmentation. Immediately after an implant is placed in native bone, the implant surface is in direct contact with bone and is covered by blood forming a blood clot. Within 4 days, granulation tissue containing mesenchymal cells, matrix components and newly formed vascular structures partly replaces the blood clot. One week after implantation, the granulation tissue is replaced by provisional matrix rich in vessels, mesenchymal cells and fibers. At the same time, contact osteogenesis occurs presenting as direct contact between the implant and the newly formed woven bone. After two weeks, osteogenesis continues to occur and it also takes place in areas at a distance from the implant surface. Later on, the woven bone matures and bone trabeculae are reinforced with lamellar bone allowing for the development of load-bearing bone after 6-8 weeks.17 The healing process described above is called osseointegration and is defined as “a direct, structural and functional connection between ordered, living bone and the surface of a load-carrying implant fixture”.18 Bone regeneration and osseointegration at

4 previously grafted sites is also predictable.19 In a dog model, Wetzel et al19 showed that osseointegration occurs after implant placement in sinus grafted with different bone graft materials. Even though bone graft particles may be around the implant, only newly-formed bone is in contact with the implant surface, as seen in human biopsies after removal of disease-free implants 6 months and 4 and 8 years after placement in grafted sinuses.20-22

Even though osseointegration is not a concern, only a few studies reported data on the dimensional stability of the augmented bone after implant placement. In a prospective multi-center study of 2,685 implants by Spray et al a mean buccal bone thickness reduction of 0.7mm was reported between implant placement and abutment connection. The healing time interval for maxillary implants was 6-8 months and 3-4 months for mandibular implants. In this trial the importance of the initial buccal bone thickness was pointed out, showing that sites with initial buccal bone thickness of 1.8mm or more were more likely to heal with bone apposition.23

Cardaropoli et al followed up implants replacing single maxillary incisors in a two stage procedure after post-extraction bone healing of 6 months. At abutment connection buccal and lingual bone height loss corresponded to 0.7-1.3mm and buccal bone thickness loss corresponded to 0.4mm.24 These observations suggest that initial bone thickness at implant placement may be a determinant of buccal bone remodeling after implant placement.

Buccal bone loss is not prevented after extraction and immediate implant placement. Chen et al highlighted the importance of the gap between the implant

5 surface and the buccal bone wall by reporting 48%, 15.8% and 20% buccal bone thickness resorption in sites treated without bone graft, with bone graft alone and with bone graft and membrane respectively. 25 Furthermore, Ferrous et al emphasized the effect of the initial buccal wall thickness on buccal bone remodeling with buccal bone walls >1mm thick initially losing less bone thickness.26 These observations suggest that initial bone thickness at implant placement may be a determinant of buccal bone remodeling after immediate implant placement.

Initial soft tissue thickness may also be related to changes in buccal bone remodeling. “Periodontal biotype” was first introduced by Siebert & Lindhe in an effort to describe gingival thickness.27 Gingiva that was ≤ 1 mm thick was classified as having “thin-scalloped” periodontal biotype and gingiva that was >1.1 mm thick was classified as having “thick-flat” periodontal biotype. In a systematic review by

Zweers et al,28 periodontal biotype is strongly associated with bone thickness around teeth. Furthermore, in a prospective study by Chappuis et al,29 buccal bone thickness and gingival biotype were strongly correlated 10 years after placement of implants in the esthetic area.

The importance of the initial soft tissue thickness in clinical outcomes has been pointed out in a systematic review by Cosyn et al30 recommending immediate implant placement in sites with intact buccal bone wall and thick biotype by means of flapless surgery in order to minimize the risk of midbuccal recession of > 1 mm.

The superior handling properties of the thick flaps are shown by Burkhart & Lang in a prospective clinical trial evaluating the effect of flap tension on primary wound

6 closure after implant placement in humans. They found that in sites with higher tension during wound closure, flaps that were > 1mm thick manifested wound dehiscences less frequently. 31 Furthermore, Hwang & Wang found that flap thickness of > 1.1 mm is a predictor of complete root coverage irrespective of the procedure type32 Therefore, initial soft tissue thickness may affect the outcomes of flap procedures.

Handling of the flap may affect the blood supply it provides to underlying buccal bone, therefore surgical trauma, including flap elevation, vertical incisions and suturing may disrupt the blood circulation at the area and impede the nutrient supply to the bone. Fickl et al showed in a dog model that flap elevation results in crestal bone loss, which is less pronounced after a partial thickness flap is elevated leaving the periosteum and some connective tissue attached to the bone.33 Wood et al concluded that the crestal bone loss after flap elevation is associated with the thickness of the bone with thin bone undergoing more remodeling.34 Flapless immediate implant placement results in 0.51mm less buccal bone loss compared to a flapped immediate implant placement in the dog model.35 Therefore, the different flap manipulations altering the blood supply provided by the flap seems to affect the buccal bone remodeling.

Laser Doppler Flowmetry is a non-invasive technique for detecting changes in the blood flux. It has been extensively used in plastic surgery36 and it started being used for evaluation of oral and gingival blood flow by Baab et al in the

1980s.37, 38 Immediately after surgery the blood perfusion is characterized by an

7 ischemic response due to surgical trauma and the use of local anesthetic with vasoconstrictor.36, 39-41 After periodontal flap procedures the buccal flap responds with an increase in blood perfusion until the 7th day followed by a decrease to pre- operative levels by the 15th day.36 After extraction and alveolar ridge preservation the buccal flap blood flow follows an ischemia-reperfusion model with hyperemia persisting for up to 1 month.41 Laser Doppler flowmetry has been also used for evaluation of bone blood perfusion. In a prospective study in humans, bone blood perfusion at the implant site has been positively correlated with implant stability.42

Implant stability is a measure of osseointegration and is classified as primary and secondary. Primary stability derives from the mechanical engagement of the osteotomy walls by the implant. Secondary stability is characterized by bone apposition on the implant surface indicating the time of loading.43 Methods assessing implant stability include Periotest® and resonance frequency analysis.

Periotest® is an electronically controlled mechanical tapping device which measures the damping characteristics of tissues around implants.43 However, due to repeated tapping movements of the device on the implant, the bone-to-implant interface may experience potential damage especially in low quality bone.44

Resonance frequency analysis, as expressed in implant stability quotient values, is a non-invasive method that measures the changes in the stiffness of the bone-to- implant interface with higher values representing higher degree of osseointegration.

Implant stability can be linked to bone changes as implant stability quotient values

90 and 150 days after implant placement in healed sites are higher than after

8 immediate implant placement. 45 When implants are placed in regenerated sites, the implant stability quotient values increase similarly to stability values of implants placed in pristine bone.46 However, the implant stability values are higher when healing of regenerated bone before implant placement is allowed for 12 months compared to 6 months.46 Therefore, bone remodeling during early phases of healing may be represented by implant stability values as measured by resonance frequency analysis.

Even though the effect of the aforementioned parameters on bone remodeling has been previously studied, there are no reports evaluating buccal flap blood perfusion, buccal bone remodeling and implant stability comparing grafted and non-grafted sites in the same cohort. The study of the influence of these parameters along with initial buccal bone thickness and tissue biotype may provide important information on early wound healing after implant placement in grafted and non-grafted esthetic zone sites.

Purpose of the study

This study aims to determine the rate of recovery from surgical trauma through blood perfusion following implant placement surgery.

9 Working hypothesis

The working hypothesis is that the rate of recovery is different between implant sites in which previous/simultaneous grafting is performed and implant sites in which implant was placed into pristine host native bone.

Specific aims for this thesis

1. Describe clinical peri-implant wound healing parameters at anatomically similar grafted and non-grafted sites.

2. Determine the recovery of flap blood perfusion rate following implant placement surgery at grafted and non-grafted sites.

3. Compare stability of implant fixture placed into grafted and non-grafted sites during early phases of healing (prior to mechanical loading).

4. Determine changes in peri-implant buccal bone thickness at grafted and non- grafted sites prior to mechanical loading.

10 References

1. Atwood DA, Coy WA. Clinical, cephalometric, and densitometric study of reduction of residual ridges. J Prosthet Dent 1971;26:280-295. 2. Trombelli L, Farina R, Marzola A, Bozzi L, Liljenberg B, Lindhe J. Modeling and remodeling of human extraction sockets. J Clin Periodontol 2008;35:630- 639. 3. Araujo MG, Lindhe J. Dimensional ridge alterations following tooth extraction. An experimental study in the dog. J Clin Periodontol 2005;32:212- 218. 4. 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. 5. Van der Weijden F, Dell'Acqua F, Slot DE. Alveolar bone dimensional changes of post-extraction sockets in humans: a systematic review. J Clin Periodontol 2009;36:1048-1058. 6. 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. 7. Moya-Villaescusa MJ, Sanchez-Perez A. Measurement of ridge alterations following tooth removal: a radiographic study in humans. Clin Oral Implants Res 2010;21:237-242. 8. Chappuis V, Engel O, Reyes M, Shahim K, Nolte LP, Buser D. Ridge alterations post-extraction in the esthetic zone: a 3D analysis with CBCT. J Dent Res 2013;92:195S-201S. 9. Huynh-Ba G, Pjetursson BE, Sanz M, et al. Analysis of the socket bone wall dimensions in the upper maxilla in relation to immediate implant placement. Clin Oral Implants Res 2010;21:37-42. 10. Chappuis V, Araujo MG, Buser D. Clinical relevance of dimensional bone and soft tissue alterations post-extraction in esthetic sites. Periodontol 2000 2017;73:73-83. 11. 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. J Clin Periodontol 2008;35:356-363. 12. Benic GI, Hammerle CH. Horizontal bone augmentation by means of guided bone regeneration. Periodontol 2000 2014;66:13-40. 13. Jung RE, Ioannidis A, Hammerle CHF, Thoma DS. Alveolar ridge preservation in the esthetic zone. Periodontol 2000 2018;77:165-175. 14. Nyman S, Lang NP, Buser D, Bragger U. Bone regeneration adjacent to titanium dental implants using guided tissue regeneration: a report of two cases. Int J Oral Maxillofac Implants 1990;5:9-14. 15. Ten Heggeler JM, Slot DE, Van der Weijden GA. Effect of socket preservation therapies following tooth extraction in non-molar regions in humans: a systematic review. Clin Oral Implants Res 2011;22:779-788. 11 16. Horvath A, Mardas N, Mezzomo LA, Needleman IG, Donos N. Alveolar ridge preservation. A systematic review. Clin Oral Investig 2013;17:341-363. 17. Salvi GE, Bosshardt DD, Lang NP, et al. Temporal sequence of hard and soft tissue healing around titanium dental implants. Periodontol 2000 2015;68:135-152. 18. Schenk RK, Buser D. Osseointegration: a reality. Periodontol 2000 1998;17:22-35. 19. Wetzel AC, Stich H, Caffesse RG. Bone apposition onto oral implants in the sinus area filled with different grafting materials. A histological study in beagle dogs. Clin Oral Implants Res 1995;6:155-163. 20. Valentini P, Abensur D, Densari D, Graziani JN, Hammerle C. Histological evaluation of Bio-Oss in a 2-stage sinus floor elevation and implantation procedure. A human case report. Clin Oral Implants Res 1998;9:59-64. 21. Scarano A, Pecora G, Piattelli M, Piattelli A. Osseointegration in a sinus augmented with bovine porous bone mineral: histological results in an implant retrieved 4 years after insertion. A case report. J Periodontol 2004;75:1161-1166. 22. Degidi M, Piattelli A, Perrotti V, Iezzi G. Histologic and histomorphometric evaluation of an implant retrieved 8 years after insertion in a sinus augmented with anorganic bovine bone and anorganic bovine matrix associated with a cell-binding peptide: a case report. Int J Periodontics Restorative Dent 2012;32:451-457. 23. Spray JR, Black CG, Morris HF, Ochi S. The influence of bone thickness on facial marginal bone response: stage 1 placement through stage 2 uncovering. Ann Periodontol 2000;5:119-128. 24. Cardaropoli G, Lekholm U, Wennstrom JL. Tissue alterations at implant- supported single-tooth replacements: a 1-year prospective clinical study. Clin Oral Implants Res 2006;17:165-171. 25. Chen ST, Darby IB, Reynolds EC. A prospective clinical study of non- submerged immediate implants: clinical outcomes and esthetic results. Clin Oral Implants Res 2007;18:552-562. 26. Ferrus J, Cecchinato D, Pjetursson EB, Lang NP, Sanz M, Lindhe J. Factors influencing ridge alterations following immediate implant placement into extraction sockets. Clin Oral Implants Res 2010;21:22-29. 27. Lindhe J, Lang NP, Berglundh T, Giannobile WV, Sanz M. Clinical periodontology and implant dentistry. Chichester, West Sussex ; Ames, Iowa: John Wiley and Sons, Inc.; 2015: p. 28. Zweers J, Thomas RZ, Slot DE, Weisgold AS, Van der Weijden FG. Characteristics of periodontal biotype, its dimensions, associations and prevalence: a systematic review. J Clin Periodontol 2014;41:958-971. 29. Chappuis V, Rahman L, Buser R, Janner SFM, Belser UC, Buser D. Effectiveness of Contour Augmentation with Guided Bone Regeneration: 10-Year Results. J Dent Res 2018;97:266-274.

12 30. Cosyn J, Hooghe N, De Bruyn H. A systematic review on the frequency of advanced recession following single immediate implant treatment. J Clin Periodontol 2012;39:582-589. 31. Burkhardt R, Lang NP. Role of flap tension in primary wound closure of mucoperiosteal flaps: a prospective cohort study. Clin Oral Implants Res 2010;21:50-54. 32. Huang LH, Neiva RE, Wang HL. Factors affecting the outcomes of coronally advanced flap root coverage procedure. J Periodontol 2005;76:1729-1734. 33. Fickl S, Kebschull M, Schupbach P, Zuhr O, Schlagenhauf U, Hurzeler MB. Bone loss after full-thickness and partial-thickness flap elevation. J Clin Periodontol 2011;38:157-162. 34. Wood DL, Hoag PM, Donnenfeld OW, Rosenfeld LD. Alveolar crest reduction following full and partial thickness flaps. J Periodontol 1972;43:141-144. 35. Blanco J, Linares A, Perez J, Munoz F. Ridge alterations following flapless immediate implant placement with or without immediate loading. Part II: a histometric study in the Beagle dog. J Clin Periodontol 2011;38:762-770. 36. 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. J Periodontal Res 2005;40:129-137. 37. Baab DA, Oberg A, Lundstrom A. Gingival blood flow and temperature changes in young humans with a history of periodontitis. Arch Oral Biol 1990;35:95-101. 38. Baab DA, Oberg PA. Laser Doppler measurement of gingival blood flow in dogs with increasing and decreasing inflammation. Arch Oral Biol 1987;32:551-555. 39. Retzepi M, Tonetti M, Donos N. Gingival blood flow changes following periodontal access flap surgery using laser Doppler flowmetry. J Clin Periodontol 2007;34:437-443. 40. 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. J Clin Periodontol 2007;34:903- 911. 41. Alssum L, Eubank TD, Roy S, et al. Gingival Perfusion and Tissue Biomarkers During Early Healing of Postextraction Regenerative Procedures: A Prospective Case Series. J Periodontol 2017;88:1163-1172. 42. Kokovic V, Krsljak E, Andric M, et al. Correlation of bone vascularity in the posterior mandible and subsequent implant stability: a preliminary study. Implant Dent 2014;23:200-205. 43. Atsumi M, Park SH, Wang HL. Methods used to assess implant stability: current status. Int J Oral Maxillofac Implants 2007;22:743-754. 44. Seong WJ, Conrad HJ, Hinrichs JE. Potential damage to bone-implant interface when measuring initial implant stability. J Periodontol 2009;80:1868-1874.

13 45. Gehrke SA, da Silva Neto UT, Rossetti PH, Watinaga SE, Giro G, Shibli JA. Stability of implants placed in fresh sockets versus healed alveolar sites: Early findings. Clin Oral Implants Res 2016;27:577-582. 46. Deli G, Petrone V, De Risi V, Tadic D, Zafiropoulos GG. Longitudinal implant stability measurements based on resonance frequency analysis after placement in healed or regenerated bone. J Oral Implantol 2014;40:438-447.

14 CHAPTER II

Maxillary Buccal Bone Dimensional Measurement by CBCT:

Development of a method to determine dimensional changes following dental

implant placement surgery

Abstract

Objective: Cone Beam Computed Tomography (CBCT) is a useful tool for three dimensional evaluation of hard tissue contours prior to and following implant- related surgeries. The purpose of this study was to develop a reliable method to determine volumetric and linear changes within buccal bone following implant placement surgeries. Results were compared with previously used technique to evaluate regenerated bone volume within similar anatomical location.

Materials and Methods: CBCT documentation of the edentulous anterior maxilla following bone regeneration (socket preservation [SP] and guided bone regeneration [GBR]) and/or implant placement surgeries was completed during first post-operative week and repeated following 4 months of healing. Buccolingual, linear and volumetric measurements targeting buccal bone thickness were

15 performed by using standard marks embedded into surgical guides or by using adjacent teeth to define the area of interest while comparing two CBCTs obtained from the same subject. In CBCTs taken after implant placement, peri-implant buccal bone changes along the length of the implant were calculated by using a software.

Buccal bone grey values were selected based on soft tissue and bone grey values after automatic superimposition of images. T-test or Wilcoxon-Man-Whitney test, as appropriate, was used to analyze all the data. Spearmann’s correlation coefficient was used for correlation analyses. The advantages and disadvantages of both techniques were discussed.

Results: 13 CBCT records following post-tooth extraction and bone regeneration procedures and 20 CBCT records following implant placement surgery were prospectively collected. Linear distance measurements from the surgical stent to ridge revealed - 0.75 ± 0.24 mm buccal bone loss in SP and - 0.97 ± 0.16 mm buccal bone loss in GBR group, respectively. Mean 3-D bone volume changes, determined by using adjacent teeth as standardization points and subtracting buccal half of the ridge for calculations, revealed volumetric bone loss of – 0.22 ± 0.08 cm3 and – 0.33

± 0.07 cm3 in SP and GBR groups, respectively (p=0.05 between groups). The software used to determine buccal bone thickness at various vertical levels along the newly placed dental implant revealed a mean buccal bone loss of 0.1±0.008 mm

(0.06-0.1 mm) and 0.3±0.06 mm (0.1-0.5 mm) in non-grafted and grafted sites, respectively (p<0.01, between groups) following 4 months of healing.

Conclusion: Buccal bone dimensional changes after post-extraction bone regeneration procedures and/or following implant placement can be determined by using linear and volumetric changes based on a standard object localized at the buccal aspect of the ridge or by using dental and anatomical landmarks. However, computerized volumetric evaluation and automatic selection of the areas of interest following CBCT image superimposition allow for better evaluation of bone changes as long as radiation exposure and dental implant related artifacts can be controlled.

17 Introduction

Alveolar bone loss after tooth extraction is well documented.47, 48 Bone regeneration techniques are employed to preserve and/or augment the existing bone after tooth extraction. Depending on the number of socket walls left after extraction, bone graft material is placed in the socket with or without barrier membrane coverage and/or addition of biological mediators. According to a systemic review and meta analysis by Vignoletti et al,49 extraction and bone regeneration results in less alveolar bone height and width loss of 1.47mm and

1.83mm respectively compared to extraction alone. Research included into this and similar systematic reviews use a great variety of methods to assess the alveolar bone changes; among those, impressions and stone casts, reentry surgical measurements and clinical, radiographic and histological analyses have been reported1, 34, 48, 50. Routine dental radiographs, such as lateral cephalographs and panoramic x-rays, cannot be effectively used to evaluate alveolar ridge bone dimensions, since they are 2-dimensional representation of a 3-dimensional object of interest. With the introduction of computed tomography into dentistry, the 3- dimensional evaluation of the ridge prior to and after various augmentation surgeries is part of current standard care.51 This technique allows the acquisition of cross sections that are perpendicular to the curvature of the jaw bones. The advantage of a high spacial resolution along with a low acquisition time and radiation dose is significant in dental practice. In addition, Cone Beam Computed

18 Tomography (CBCT) can be effectively used in clinical research as long as some standardization tools are developed.

Literature on CBCT documentation of various dental/oral surgeries related to hard tissue augmentation already exists. 15, 41, 49 To list a few, Fiorelini et al performed linear measurements on CBCTs 4 months after extraction and recombinant human bone morphogenetic protein-2 (rhBMP-2) delivery in a bioabsorbable collagen sponge in order to assess the outcome of post-extraction bone regeneration.52 Brownfield and Weltman used CBCT to evaluate 4-month healing of extraction sockets with and without bone graft material.53 Aimetti et al used linear and volumetric measurements on CBCTs to determine bone dimensional changes of compromised extraction sockets filled with bone graft material covered with membrane after 12 months of healing.54 Fiorelini et al52 used ridge-based lines to calculate changes, whereas Brownfield and Weltman53 measured distances from a radiopaque reference point on a stent that the patients wore during scanning. In both studies, the measurements were done separately on the baseline and follow-up

CBCT. On the contrary, Aimetti et al54 used specific landmarks to accurately superimpose the baseline and follow-up CBCT images and then performed measurements in the resulting superimposed images.

When documenting an edentulous site for future implant supported restorations, as well as when evaluating long-term stability of peri-implant tissue contours, measurement of linear changes compared to volumetric changes have significant limitations. Veltri et al55 and Schropp et al56 evaluated buccal and crestal

19 bone changes of implants in function for an average of 9 and 10 years respectively.

Both studies superimposed CBCT images taken after implant placement with the

CBCT images taken at follow-up appointment using the implant or fixed bone structures as reference points. The buccal bone measurements were expressed as bone thickness values in mm at specific distances from the implant platform55, 56 or as area volume in the most coronal 2mm of the implant.55

There is significant interest in studying factors affecting buccal bone integrity in the esthetic zone. We aimed to modify existing techniques to obtain reliable readings specific for buccal bone thickness from repeated and superimposed CBCTs without increasing radiation exposure time and controlling artifacts. Thus, the purpose of this study was to develop a reliable method to determine volumetric and linear changes within buccal bone following implant placement surgeries. Data was compared with previously used technique to evaluate regenerated bone volume within similar anatomical location.

Materials and Methods

Study design:

The study was a prospective observational clinical trial. Patients treatment planned for extraction and/or bone regeneration (SP or GBR) and implant placement at a maxillary non-molar single site were recruited in two cohorts. The first cohort included patients receiving extraction and bone regeneration before

20 implant placement and the second cohort included patients receiving single implant.

A CBCT was taken immediately after surgery and at 4 months. Both study protocols

(protocol #2014H0150 for extraction and bone regeneration surgery and

#2015H0125 for implant placement surgery), data collection and informed consent forms were approved by the Institutional Review Board of the Ohio State University.

Subject population:

Patients referred to the Graduate Periodontics Clinic at OSU for implant placement for a single maxillary non-molar implant with intact adjacent teeth were recruited. Eligibility criteria were as follows: non-smokers, aged 18-75 years treatment planned to receive single implant in maxillary non-molar region; implant site bounded by adjacent teeth; no systemic diseases/conditions affecting periodontal health or disease; non-pregnant and non-lactating; no untreated periodontal disease; able and willing to provide informed consent for surgery and study. Exit criteria were as follows: voluntary withdrawal, non-compliance with study protocol, no longer meeting eligibility criteria (changes in surgical procedures, development of systemic/oral disease). All subjects signed informed consent form prior to surgical procedures.

CBCT scan, image acquisition and process:

CBCT images were obtained using i-CAT system (Imaging Sciences

International, Hatfield, PA, USA). The CBCT settings were 8 × 8 cm Field of View,

21 14.7 seconds exposure time and 0.2 mm voxel size. The CBCTs were taken 3-9 days after the bone regeneration or implant surgery and at 4 months. This protocol was formulated by an oral radiologist for achieving radiation reduction while obtaining detailed images of the area of interest. The patients receiving extraction and bone regeneration were scanned while wearing a stent with a radiopaque insert (gutta- percha point). This insert was placed on the midbuccal of the surgical area and was used as a reference point for measurements.

Data were saved in hard discs and were converted to DICOM (Digital Imaging and Communications in Medicine) format. CBCT image distortion was tested by measuring a stable structure in both CBCTs of each patient. The measurements were compared using paired t-test. Then, the DICOM files of sites receiving extraction and bone regeneration were imported to OsiriX (OsiriX Lite v.7.0.2 Pixmeo, Geneva,

Switzerland) software for analysis. The DICOM files of sites receiving single implant were imported to MevisLab (MeVis Medical Solutions AG, Fraunhofer MEVIS,

Bremen & Lübeck, Germany) software for analysis.

CBCT image analysis of sites receiving extraction and bone regeneration:

Buccal-Lingual (BL) measurements:

BL measurement represents the buccolingual width of the ridge at the most coronal part of the ridge (Figure 1). 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

22 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.) as seen in Figure 1. This measurement was recorded in mm and already published (Alssum et al 2017).

Linear measurements:

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

(Gutta Purcha placed into surgical guide) and the buccal plate (Figure 2). 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) was dropped from the reference point to the line A (modified from Lee et al.57). Line B represents the distance from the buccal plate, and can indicate bone dimensional changes on the buccal aspect of the ridge (Figure 1). 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) and related data was already published (Alssum et al 2017).

3-dimensional measurements:

The 3-D measurement represents the volume change in the grafted site. Due to difficulty in separating 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

23 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) gave the dimensional change (ΔD) at the buccal, palatal and coronal aspect of the ridge.

Starting from the root canal of the adjacent teeth 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 3). Ridge dimensional change was calculated as follows: ΔD=

D1- D0 This measurement was recorded in cubic centimeters (cm3) and related data was already published (Alssum et al 2017).

CBCT image analysis of sites receiving single implant:

After DICOM files were imported in MATLAB (MathWorks, Natick, MA, USA) software, the maxillary bone and teeth were segmented from each image using a region growing based semi-automatic segmentation method. The images were then imported in MevisLab software and were registered using rigid registration

(superimposition) with minimization of mean square error which involves translations and rotations in order to minimize the distance between the grey values of two images. After each step of translation and/or rotation (iteration), mean squared error (MSE) function is computed. Iterations continue until the MSE

24 is minimized as shown in Figure 5. Figure 6 shows the images before and after the registration.

After the images were registered, a 3-D rectangular parallelepiped-shaped

Region of Interest (ROI) including the area of the implant and part of the adjacent teeth was selected as shown in Figure 7. This allowed for viewing of only the ROI in another window in sagittal, coronal and axial plane. The images were rotated so that the implant was perpendicularly oriented in the coronal and sagittal cross sections.

From this window, another 3-D rectangular parallelepiped-shaped ROI (implant

ROI) was selected including only the implant in the coronal view. In the sagittal and axial views, the implant ROI included the buccal half of the implant and was extended buccally up to 10mm from the center of the implant for inclusion of the buccal bone of the implant as shown in Figure 8. The implant ROI was projected in another window for comparison of buccal bone thickness changes. This window included all axial images (every 0.2mm) of the implant in the first and second CBCT and the registered images as shown in Figure 9.

The buccal bone’s grey values were automatically identified by the software according to upper and lower grey value thresholds. The thresholds were selected manually from soft tissue and bone areas in each CBCT to control for grey value differences between the two CBCTs. The soft tissue control area was selected from the gingiva palatal to the tooth contralateral of the implant as shown in Figure 10.

The bone control area was selected from the cortical bone of the lower border of the mandible in the mental region as shown in Figure 11. The highest grey value of the

25 soft tissue control area plus one grey value was selected as the lower grey value threshold in order to exclude the soft tissue and air grey values of the implant ROI from the buccal bone area. The highest grey value of the bone control area was selected as the upper threshold in order to include cancellous and cortical bone and exclude the implant grey values of the implant ROI from the buccal bone area.

The superimposed buccal bone areas were exported as DICOM files to

MATLAB (MathWorks, Natick, MA, USA) software where the calculation of the buccal bone thickness in the first CBCT and the difference in thickness in the superimposed images from the first and second CBCT was done.

Buccal bone thickness in the first CBCT was calculated using the segmented buccal bone region pixels shown as the red area in Figure 5. Starting from the first pixel coordinate in horizontal axis (left to right), maximum difference between pixel coordinates in vertical axis covered by the segmented area was calculated. Then the pixel difference was multiplied by the distance between pixels (0.2 mm) and thickness was calculated for the first column of the ROI. This calculation was done for each column in the implant ROI. Difference in bone thickness between the first and second CBCTs was calculated using the difference between the vertical coordinates of the pixels farthest from the implant and covered by the segmented areas for the first (red) and second (blue) CBCTs.

After these heat map-like tables of initial buccal bone thickness in the 1st CBCT

(Figure 12) and buccal bone thickness difference between 1st and 2nd CBCT (figure

26 13) along the width (length of diameter) and length of the implant were completed, only measurements in the middle third of the implant width were selected for statistical analysis. As shown in Figure 7, areas that were as wide as 1/3 of the implant diameter and as long as 2mm apicocoronally were selected and the average of the values in the selected boxes was used as the initial thickness of the buccal bone and bone difference in this specific length interval. These measurements were performed along the length of the entire implant.

Registration of images, software development, selection of ROI and implant

ROI and calculation of initial bone thickness and difference in bone thickness between the two CBCTs was done by a medical imaging specialist (Dr. Mutlu

Demirer). Image calibration related to superimposition for each subject was completed by using specific software. Selection of control areas was done by a clinician (Dr. Vrisiis Kofina). The clinician was trained in selecting anatomical landmarks that would work as control. Soft and hard tissue control areas were selected visually after adjusting the brightness of the image for easier visualization.

Statistical analysis

T-test or Wilcoxon-Man-Whitney test, as appropriate, was used to analyze data.

27 Results

Study population related demographics and wound healing parameters for the two separate cohorts were provided in already published work (Alssum et al

2017) and in Chapter III of this thesis.

Linear measurements revealed a mean change (ΔL) of -0.75 ± 0.24 mm

(ranging from -1.83 to 0.02 mm) for SP group, 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) (Alssum et al 2017).

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) (Alssum et al 2017).

Data obtained by superimposition of CBCTs following implant placement surgery revealed a mean loss of 0.1±0.008 mm (0.06-0.1 mm) and 0.3±0.06 mm

(0.1-0.5 mm) in non-grafted and grafted sites, respectively (p<0.01, between groups). The most pronounced bone thickness loss was recorded at 6-8 mm along the implant length (p=0.02, within grafted group).

At the most coronal 2 mm along the implant length, where the flap was elevated, grafted sites with initial buccal bone thickness ≥1.5 mm lost more bone thickness compared to sites with initial buccal bone thickness <1.5 mm (p<0.01). At

28 4 months, buccal bone thickness was 1.42±0.09mm (1.24-1.5 mm) and 1.46±0.05 mm (1.2-1.8mm) in non-grafted and grafted group, respectively (p>0.05).

Discussion

Dental implants have been a popular treatment choice for replacing missing teeth. For a predictably biologic, functional and esthetic implant outcome, buccal and lingual bone thickness of at least 1mm is required around the implant.58 In order to achieve appropriate bone dimensions after extraction, bone preservation and augmentation procedures are performed. Tools to evaluate outcomes of these regenerative procedures are limited. The purpose of this study was to develop a reliable method to determine volumetric and linear changes within buccal bone following implant placement surgery. Methodology based on CBCT analysis was compared with previously used technique to evaluate regenerated bone volume within similar anatomical location.

For evaluation of bone augmented sites, buccolingual, linear and volumetric

CBCT measurements were performed in image sets of the baseline and 4-month- follow-up CBCTs separately. For evaluation of the buccal bone following implant placement, previously used protocol was improved so that baseline and 4-month- follow-up CBCT were superimposed and then automatic controlled linear measurements in every 0.2mm axial cross sections were performed to evaluate buccal bone area thickness changes.

29 In the bone regeneration part of our study, buccolingual measurements were done in the midsagittal cross section of the edentulous regenerated area in order to evaluate the feasibility of implant placement following 4-month healing of extraction and bone regeneration. Even though buccolingual ridge measurements in the midsagittal cross section of the augmented ridge may be adequate for treatment planning purposes, they cannot appreciate the magnitude of changes in the buccal area of the socket or the entire grafted area. For this reason, linear measurements focusing only on the buccal area and volumetric measurements in the entire grafted area were done. For buccal bone changes, the patients were scanned wearing a stent with a radiopaque insert and linear measurements were performed having a radiopaque insert as a reference point. The radiopaque insert was placed in the middle of the edentulous area mesiodistally, therefore only one sagittal cross section was evaluated. Although surgical guides with standard radiopaque markers embedded in them are accepted standardization tools in oral radiology, this one point approach for linear measurements within a 3-D model significantly limits the area of interest. In addition, although the guide and the location of the radiopaque marker in the guide are same for both CBCT documentations, the rate and the exact location of buccal plate remodeling negatively affect repeating the linear measurements from the same region.

The volumetric measurements of the entire edentulous area were easier to standardize compared to linear measurements. The root canals of the adjacent teeth were used as landmarks for these measurements. Thus, the entire edentulous area

30 was included in the analysis. This, however, did not necessarily represent only the regenerated bone area. A possible solution would be the acquisition of a CBCT before extraction to facilitate the area selection of only the root area for more precise linear and volumetric measurements after superimposition of CBCTs.

However, this would increase the radiation dose to the patient. Another possible solution would be to identify the grey values of the bone graft material in order to isolate the grafted area, as performed in Aimetti et al’s study.54 This, nevertheless, is challenging and usually has to be done manually, because the grey values of the bone graft used (generally of human or bovine origin) are similar to native bone.

Thus, changes in volumetric dimensions were not easily detected despite rigid inclusion criteria of a single tooth edentulous area surrounded with teeth and similar anatomical locations.

In the implant part of our study, buccal bone thickness changes were measured after automatic registration of the baseline and 4-month-follow-up CBCT.

In the superimposed images, the implant itself served as a reference to allow for isolation of its buccal aspect. From the isolated implant buccal aspect, the identification of the grey values of the buccal bone was performed automatically by the software according to control soft and hard tissue grey value thresholds selected in the baseline and follow-up CBCT. The selection of control thresholds separately from each CBCT was necessary, since differences in grey value intensity were found between CBCTs of the same patient during the analysis. The palatal gingiva of the tooth contralateral of the implant site served as the control area for the soft tissue

31 threshold. This area is considered stable, because it was not surgically manipulated.

The cortical bone of the lower border of the mandible served as the control area of cortical bone, since its grey values are representative of high density cortical bone.

By using the control thresholds, we managed to select only the cancellous and cortical buccal bone of the implant excluding the soft tissues and implant itself from the automatic selection of grey values. This was performed in all axial cross sections

(every 0.2mm) of the implant allowing monitoring of buccal bone thickness changes all along the buccal aspect of the implant. For the present analysis, only the area that corresponded to the middle third of the implant diameter mesiodistally was selected. This area was separated in 2mm zones apicocoronally in order to localize bone changes along the buccal aspect of the implant. Presence of dental implant with known diameter and length on CBCT tremendously helps with standardization of region of interest in CBCTs obtained from the same individual at different time points.

The development of the CBCT analysis used for the implant part of this study was a natural step forward from the previous analysis of the CBCTs after bone regeneration procedures. The need for greater standardization led to the use of automatic superimposition of CBCTs, selection of the fixed structure (implant) as a reference point in order to isolate the study area and automatic selection of bone grey values according to control soft and hard tissue thresholds. The manual selection of areas was only limited to the selection of the control soft and hard tissue areas. However, there were certain limitations with this analysis. The presence of

32 the implant metal structure produces beam hardening artefacts appearing as cupping or streaking artefacts.59 According to Benic et al60, bone grey values appear increased or decreased depending on the location of the bone along the mesiodistal aspect of the implant. In order to minimize the effect of the artifacts on the analysis and have uniform bone grey values, only the middle part of the implant mesiodistally was selected. Another limitation related to the artefact produced by the implant is the difficulty in detecting thin bone in proximity of the implant.

According to a review by Benic et al61, bone detection was more accurate when the thickness of the cortical bone around the implant was > 0.5mm. In the present study, initial buccal bone thickness of >1mm was detected clinically at the time of implant placement. However, since bone loss was detected at 4 months in all sites, the 4-month-measurements may be subject to error due to the effect of the artefact on thinner buccal bone. Furthermore, voxel size < 0.3mm is related to better resolution and detection of thin cortical bone around implants.62, 63 In order to overcome this limitation, our CBCTs were done with a small voxel size (0.2mm).

Conclusion

In conclusion, CBCT is a useful tool for evaluation of bone changes after implant-related surgeries. This is the first study that measured bone changes after 4 months of healing at regenerated bone single-tooth edentulous areas and single implants with CBCT technology. The volumetric evaluation, automatic superimposition of images and automatic selection of bone grey values based on

33 control soft and hard tissue grey value thresholds was necessary for better standardization of the technique in an effort to evaluate a specific region of interest and minimize human error. Bone dimensional changes after post-extraction bone regeneration do not prevent implant placement and continue to a minimal extent even after the implant is placed.

Figure 1. Buccolingual 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.

34 Figure 2. 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.

Figure 3. 3-D Object

Figure 4. 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)

Figure 5. Minimization of mean squared error for image registration of subject #4

36 Figure 6. CBCT images of subject #8 immediately (blue) and 4 months (red) after implant placement #11;. Sagittal (A), coronal (B) and axial (C) view of the CBCT images before registration of two CBCTs. Sagittal ( D), coronal (E) and axial (F) view after registration of two CBCTs.

Figure 7. Region of interest (ROI); Selection (yellow box) in axial (A), coronal (B) and sagittal images (C) in the superimposed first and second CBCT of subject #23.

37 Figure 8. Magnification of selected ROI from figure 7 - Implant Region of Interest (Implant ROI); Selection (yellow box) simultaneously though synchronization of box movement in first (top) and second (bottom) sagittal (A), coronal (B) and axial (C) CBCT images of patient #23.

Figure 9. Magnification of image C of figure 8 – Buccal bone selection; Identification of buccal bone regions (pink and blue outlined regions) for first (A) and second (B) CBCT images and superimposed (C) CBCT images of patient #23.

Figure 10. Soft tissue control area; Selection (yellow box) simultaneously though synchronization of box movement in first (top) and second (bottom) sagittal (A), coronal (B) and axial (C) CBCT images of patient #23.

Figure 11. Bone control area; Selection (yellow box) simultaneously though synchronization of box movement in first (top) and second (bottom) sagittal (A), coronal (B) and axial (C) CBCT images of patient #23.

39 Figure 12. Initial peri-implant buccal bone thickness Figure 13. Peri-implant buccal bone thickness heat map of subject #19. Each cell represents the difference between first and second CBCT (CBCT2- initial buccal bone thickness in each 0.2x0.2mm area CBCT1) heat map of subject #19. Each cell represents that it corresponds to. The selected box represents the buccal bone thickness difference in each the selected cells for calculation of initial buccal bone 0.2x0.2mm area that it corresponds to. The selected thickness of the most coronal 2mm area of the box represents the selected cells for calculation of midbuccal of the implant. The mean of these cells is buccal bone thickness difference of the most coronal the reported as the initial buccal bone thickness of 2mm area of the midbuccal of the implant. The mean the selected area. of these cells is the reported as the buccal bone thickness difference of the selected area.

40 References

1. Hammerle CH, Araujo MG, Simion M, Osteology Consensus G. Evidence-based knowledge on the biology and treatment of extraction sockets. Clin Oral Implants Res 2012;23 Suppl 5:80-82. 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. 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. 4. Atwood DA, Coy WA. Clinical, cephalometric, and densitometric study of reduction of residual ridges. J Prosthet Dent 1971;26:280-295. 5. Araujo M, Linder E, Lindhe J. Effect of a xenograft on early bone formation in extraction sockets: an experimental study in dog. Clin Oral Implants Res 2009;20:1-6. 6. Wood DL, Hoag PM, Donnenfeld OW, Rosenfeld LD. Alveolar crest reduction following full and partial thickness flaps. J Periodontol 1972;43:141-144. 7. Harris D, Horner K, Grondahl K, et al. E.A.O. guidelines for the use of diagnostic imaging in implant dentistry 2011. A consensus workshop organized by the European Association for Osseointegration at the Medical University of Warsaw. Clin Oral Implants Res 2012;23:1243-1253. 8. Alssum L, Eubank TD, Roy S, et al. Gingival Perfusion and Tissue Biomarkers During Early Healing of Postextraction Regenerative Procedures: A Prospective Case Series. J Periodontol 2017;88:1163-1172. 9. Ten Heggeler JM, Slot DE, Van der Weijden GA. Effect of socket preservation therapies following tooth extraction in non-molar regions in humans: a systematic review. Clin Oral Implants Res 2011;22:779-788. 10. Fiorellini JP, Howell TH, Cochran D, et al. Randomized study evaluating recombinant human bone morphogenetic protein-2 for extraction socket augmentation. J Periodontol 2005;76:605-613. 11. 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. J Periodontol 2012;83:581-589. 12. Aimetti M, Manavella V, Corano L, Ercoli E, Bignardi C, Romano F. Three- dimensional analysis of bone remodeling following ridge augmentation of compromised extraction sockets in periodontitis patients: A randomized controlled study. Clin Oral Implants Res 2018;29:202-214. 13. Veltri M, Ekestubbe A, Abrahamsson I, Wennstrom JL. Three-Dimensional buccal bone anatomy and aesthetic outcome of single dental implants replacing maxillary incisors. Clin Oral Implants Res 2016;27:956-963. 41 14. Schropp L, Wenzel A, Spin-Neto R, Stavropoulos A. Fate of the buccal bone at implants placed early, delayed, or late after tooth extraction analyzed by cone beam CT: 10-year results from a randomized, controlled, clinical study. Clin Oral Implants Res 2015;26:492-500. 15. Leblebicioglu B, Rawal S, Mariotti A. A review of the functional and esthetic requirements for dental implants. J Am Dent Assoc 2007;138:321-329. 16. Scarfe WC, Li Z, Aboelmaaty W, Scott SA, Farman AG. Maxillofacial cone beam computed tomography: essence, elements and steps to interpretation. Aust Dent J 2012;57 Suppl 1:46-60. 17. Benic GI, Sancho-Puchades M, Jung RE, Deyhle H, Hammerle CH. In vitro assessment of artifacts induced by titanium dental implants in cone beam computed tomography. Clin Oral Implants Res 2013;24:378-383. 18. Benic GI, Elmasry M, Hammerle CH. Novel digital imaging techniques to assess the outcome in oral rehabilitation with dental implants: a narrative review. Clin Oral Implants Res 2015;26 Suppl 11:86-96. 19. Razavi T, Palmer RM, Davies J, Wilson R, Palmer PJ. Accuracy of measuring the cortical bone thickness adjacent to dental implants using cone beam computed tomography. Clin Oral Implants Res 2010;21:718-725. 20. Naitoh M, Hayashi H, Tsukamoto N, Ariji E. Labial bone assessment surrounding dental implant using cone-beam computed tomography: an in vitro study. Clin Oral Implants Res 2012;23:970-974.

42 CHAPTER III

Blood perfusion and early healing following implant placement-

A comparison between grafted and non grafted sites

Abstract

Objective: This study aimed to determine the rate of recovery from surgical trauma through blood perfusion following implant placement surgery. The working hypothesis was that the rate of recovery is different between an implant site in which previous/simultaneous grafting is performed and an implant site in which implant was placed into pristine host native bone.

(GCF) collected at baseline, 1 and 4 months from the adjacent teeth was used for future multiplex analysis of metabolism mediators. Soft tissue biopsies were

43 collected from the wound site at baseline, 9 days and 4 months and stored for future bone remodeling related gene expression analysis. Buccal flap blood perfusion using

Laser Doppler Flowmetry (LDF) was recorded immediately before and after implant placement surgery, at 3, 6, 9 days and, 1 and 4 months. Soft tissue biotype and implant stability quotient (ISQ) were determined at the time of implant placement and at 4 months. Bone thickness changes along the peri-implant buccal wall were evaluated by automatic superimposition of two Cone Beam Computed

Tomographies taken immediately after surgery and at 4 months. Descriptive statistics are reported as mean±se and percentage. Patient was chosen as unit of measurement. A linear mixed model regression analysis was used for repeated continuous measures fixed and random effects within and between groups. A random effect (intercept and slope) regression analysis was conducted to estimate the slopes of the outcome over continuous time for non-grafted and grafted groups.

For repeated measure binary outcomes, generalized estimating equations (GEEs) was used. T-test, chi-square test or Wilcoxon-Man-Whitney test, as appropriate, was used to analyze all the other non-repeated data. Spearmann’s correlation coefficient was used for correlation analyses.

44 and 6 days (p=0.04), compared to non-grafted sites (peak at 6 days; <0.01). Both groups reached baseline GCF levels by 9 days. In grafted group only, thin tissue biotype was correlated with higher wound fluid production (r=0.4, p=0.03). Blood perfusion decreased significantly immediately postoperatively in both groups

(p<0.01 within both groups). At 3 days the blood perfusion recovery was 57% and

74±2 in non-grafted and grafted sites, respectively (p<0.01 in grafted group only).

Initial buccal bone thickness for non-grafted and grafted sites was 1.52±0.05 mm

12mm along the implant length in both groups (r=0.8, p=0.03). Also, implant stability quotient was positively correlated with implant diameter in both groups

(r=0.3, p=0.04).

45 despite surgical trauma, buccal bone thickness loss is minimal at both grafted and non-grafted sites and consistent with previously reported bone thickness loss after flap elevation.

46 Introduction

Bone loss after tooth extraction is well documented.1 Several studies have been published in an effort to provide guidelines for minimizing the amount of bone loss after extraction. Currently it is well established that flapless “atraumatic” tooth extraction and alveolar ridge preservation protocols is the standard of care when the extraction site is scheduled to receive an implant. The most commonly used alveolar ridge preservation protocols are socket preservation (SP) and guided bone regeneration (GBR).12 SP is performed when all four walls of the socket are intact, whereas GBR is performed when at least one wall is partially or fully missing.

During both protocols, bone graft material is placed in the socket.12 In the case of

GBR, a membrane is placed over the bone graft to selectively allow for new bone formation.14 However, even after such protocols bone loss in not prevented.

Horizontal and vertical bone reduction in case of SP or GBR is expected to be approximately 1mm or 1-3mm, respectively less compared to extraction alone.15, 16

Regardless of the procedure, the site that experiences the most significant bone loss is the buccal bone wall of the socket.5

Successful performance of bone regeneration procedures requires surgical manipulation of the tissues to various degrees. Flap elevation, vertical and periosteal releasing incisions and suturing are often done so that the grafting materials are contained in the area. During these manipulations the blood supply of the flap is disturbed leading to obstruction of nutrient flow from the flap to the underlying bone. When the implant is placed 4-6 months after the procedure, 47 changes in the flap may be already established affecting the rate of recovery from surgical trauma as expressed by blood supply and buccal bone thickness changes. In addition, residual graft material stays incorporated into newly regenerated bone as well as at the interface of soft and hard tissue and, at the interface of implant device.20-22These changes may not be as evident in sites that did not receive alveolar ridge preservation and are therefore less manipulated.

Laser Doppler Flowmetry (LDF) is a non-invasive method to determine tissue blood perfusion. It has been used in dentistry to study recovery rate of gingival flap blood perfusion following various periodontal surgeries. For instance,

Retzepi et al al in a Laser Doppler Flowmetry (LDF) study showed that after full thickness flap elevation for chronic periodontitis treatment, the flap blood perfusion reaches baseline levels at 15 days.39 Alssum et al found that after extraction and bone regeneration surgeries, the buccal flap blood perfusion follows an ischemia- reperfusion model which remains significantly greater than baseline even 4 months after the surgery.41 The difference in recovery rate between these two LDF studies may indicate that biomaterials used for alveolar ridge preservation potentially affect blood perfusion.

The blood perfusion of the flap depends on the amount of blood vessels in the flap which is positively associated with its thickness. Tissue biotype has been pointed out as a significant factor affecting flap outcomes. Cosyn et al. showed that after extraction and immediate placement, patients with “thin-scalloped” biotype

(or gingiva) tend to have more midbuccal recession.30 Chappuis et al. found that

48 gingival biotype and buccal bone thickness are strongly correlated 10 years after implant placement in the esthetic area.29 Therefore, tissue biotype may be a determinant of recovery rate from surgical trauma after implant placement surgery.

It is well established that the response of bone to surgical trauma is bone remodeling.8, 34 However, the possible determinants of the amount of bone remodeling after implant placement surgery related surgical trauma are not well established. Since osseointegration is based on bone remodeling, the implant stability is expected to change during early healing.64 Deli et al17 showed that implants in sites that had previously received alveolar ridge preservation showed increasing ISQ values in a similar manner to implants placed in pristine bone.

However, the timing of implant placement affected the implant stability quotient values; implants placed in regenerated bone 12 months after bone grafting has superior implant stability quotient values compared to those placed after 6 months.46 This may be related to the regenerated bone response to mechanically loaded implant device as well as the possibility of an ongoing bone remodeling process at residual graft level.50, 65 Regarding changes in bone dimensions, a well documented determinant of amount of bone remodeling after extractions is initial buccal bone thickness.8 In a prospective implant study by Spray et al, initial buccal bone thickness of ≥ 1.8mm was associated with less buccal bone thickness loss between implant placement and uncovery in post-extraction healed sites.23 In cases of immediate implant placement, buccal bone walls with initial thickness of > 1mm are subject to less bone thickness loss.26 Therefore, factors such as implant stability

49 and buccal bone thickness at a previously grafted or pristine alveolar ridge site may be differentially affected during early phases of healing following implant placement surgery.

The purpose of the current study is to determine the rate of recovery from surgical trauma through flap blood perfusion following implant placement surgery.

The working hypothesis is that the rate of recovery is different between an implant site in which previous/simultaneous grafting is performed and an implant site in which implant was placed into pristine host native bone.

Materials and Methods

Study design

The study was a prospective observational clinical trial. Patients receiving a single maxillary non-molar implant with adjacent teeth were recruited. Clinical parameters and wound healing parameters were documented prior to, during, immediately after the surgery and at 3,6,9 days and 1 and 4 months postoperatively.

The study protocol and informed consent forms were approved by the Institutional

Review Board of The Ohio State University (protocol 2015H0125).

Study population

Patients referred to the Graduate Periodontics Clinic at OSU for implant placement for a single maxillary non-molar implant with adjacent teeth were

50 recruited. Eligibility criteria were as follows: non-smokers, aged 18-75 years treatment planned to receive single implant in maxillary non-molar region; implant site bounded by adjacent teeth; no systemic diseases/conditions affecting periodontal health or disease; non-pregnant and non-lactating; no untreated periodontal disease; able and willing to provide informed consent for surgery and study. Exit criteria were as follows: voluntary withdrawal, non-compliance with study protocol, no longer meeting eligibility criteria (development of systemic/oral disease). All subjects signed informed consent form prior to surgical procedures.

Surgical procedures

Implant placement surgeries were performed by 2nd and 3rd year periodontal residents in the Graduate Periodontics Clinic at OSU under direct faculty supervision. Patients were assigned to 2 groups depending on whether or not they received bone graft at any time during treatment:

Group 1: Non-grafted sites. Implants were placed in sites that did not receive bone graft at any time prior to or during the implant placement. Group 2: Grafted sites.

Implants were placed in sites that were previously grafted and/or received bone graft at the time of implant placement.

Patients were premedicated with antibiotics prior to surgery as part of routine clinical protocol. After local anesthesia was administered, a midcrestal incision was done and a full thickness flap was elevated up to 10 mm apical to the bone crest. Osteotomies were done under copious irrigation and the implant was

51 placed. If any implant threads were exposed, bone graft material (FDBA or DBBM) was placed as part of routine surgical protocol. Similarly, surgical placement followed a one- or two-stage protocol based on implant stability at the time of placement. The flaps were replaced and sutured to achieve primary closure

(covering a healing screw or adapted around a healing abutment as indicated for two or one-stage surgical protocol, respectively). After the surgery, the patients received prescriptions for analgesics (7 days) and 0.12% chlorhexidine rinse (2-3 times/day for 2 weeks) as part of clinical protocol. If bone graft was added to the surgical site at the time of the placement, antibiotics (7 days) were also prescribed to the patients as standard care. List and the amount of post-surgical medications consumed, were documented through a diary completed by patient.

Clinical measurements

The following clinical measurements were recorded:

• Probing depth (PD) at 6 sites around the two teeth adjacent to the implant

site, prior to surgery and at 4 months.

• Keratinized tissue width (KT) measured on the midbuccal of the implant site

with a UNC-15 probe prior to surgery and at 4 months.

• Tissue biotype (TB) recorded on the midbuccal aspect of the implant site 2-3

mm apical to the level of the bone crest. The tissue thickness was measured

with a non-tension wax caliper during surgery and, at 4 months. Tissue

52 biotype was classified as thick at sites with tissue thickness > 1 mm and thin

at sites with tissue thickness ≤ 1 mm (modified by Muller et al).

• Presence of plaque (P) and gingival bleeding (GB) of the treatment sextant

were recorded prior to surgery, at 3, 6 and 9 days and 1 and 4 months. A

UNC-15 probe was placed at a 45o angle to the long axis of the tooth and ran

around the teeth of the treatment sextant and the percentage of the presence

of plaque and gingival bleeding was calculated.

Clinical wound healing was scored as yes or no for each of the following wound healing parameters, according to Kloostra et al66:

• Mature wound healing, defined as complete wound closure without other

significant findings.

• Erythema, defined as increased redness compared to adjacent non-operated

sites

• Bleeding, defined as presence of spontaneous bleeding at the wound site

• Graft mobility, defined as loose subgingival bone graft material evaluated by

gentle palpation using a UNC-15 probe

• Suppuration, defined as presence of pus or discharge at the wound site

• Necrosis, defined as any visual soft and/or hard tissue necrosis at the wound

site

Clinical wound exposure and closure and hydrogen peroxide test was used to evaluate wound closure. The farthest distance between the flap margins was recorded with a UNC-15 probe as the extent of clinical wound exposure. All wound

53 healing measurements (clinical wound healing parameters, clinical wound exposure and closure and hydrogen peroxide test) were recorded at the same time as pain assessment conducted at 3, 6, 9 days and 1 and 4 months. Visual Analogue Scale

(VAS) was used to evaluate patients’ pain and discomfort after the implant placement surgery.

Wound & gingival crevicular fluid collection

Gingival crevicular fluid (GCF) was collected from the two adjacent teeth prior to surgery, at 1 and 4 months. Wound fluid was collected from the wound edges at 3, 6 and 9 days as per published protocol.11 Briefly, six sterile paper strips were inserted in the crevice or wound edges until slight resistance was felt and were left in for 30 seconds. A previously calibrated electronic volume quantification unit (Periotron 8000 ®) was used to determine the collected volume in each strip.

The samples were placed in sterile vials that were stored at -20oC for future multiplex assay to determine the concentration of various bone remodeling related mediators.

Gingival biopsies

Gingival biopsies were obtained from the implant site at the time of implant placement surgery, at 9 days and at 4 months either by using a 15C blade or a 3 mm- punch. Liquid nitrogen was used to immediately freeze the samples which were then stored in -20oC for future studies to extract RNA and investigate gene

54 expression of bone remodeling related mediators through PCR.

LDF measurements

A stone cast that was used for the fabrication of the surgical guide was obtained and a stent for the LDF measurements was fabricated from 0.06” thermo- formed material. A hole that accommodated a plastic sleeve was placed on the midbuccal of the implant site 3-4 mm from the crest. The plastic sleeve allowed for the stabilization and standardization of the LDF probe position during recordings, so that the later remains fixed, perpendicular to the gingiva and at a 0.5-1 mm distance from it.

During the LDF recordings, the patient was seated in an upright position on the dental chair and was asked to remain still for 120 seconds until the recording was completed. The measurements were done by a Periflux System 5000 PF 5010

LDPM unit (Perimed, AB, Sweden) equipped with a standard probe. Instrument calibration was done prior to measurements by means of the Perimed PF 1000

Motility Standards according to the manufacturer’s directions. The recorded signals were translated into Perfusion Units (PU) and were displayed with the Perisoft software. (PSW 2, version 2.5.5, Perimed AB). The measurements were recorded prior to and immediately after surgery, at 3, 6, 9 days and 1 and 4 months.

Resonance Frequency Analysis

For Implant Stability Quotient (ISQ) measurements, a metal insert

(SmartPeg) was screwed in the implant. ISQ was recorded through resonance 55 frequency analysis by using a probe and related device specifically designed for metal insert (Ostell AB, Gothenburg, Sweden). The measurements were repeated immediately following implant placement and at 4 months. A total of 4 measurements was recorded from the buccal, palatal, mesial and distal from the implant. Average of these 4 repeats was used for statistical analysis.

CBCT analysis

CBCT images were obtained using i-CAT system (Imaging Sciences

International, Hatfield, PA, USA). The CBCT settings were 8 × 8 cm Field of View,

14.7 seconds exposure time and 0.2 mm voxel size. The CBCTs were taken 3-9 days after the implant placement surgery and at 4 months. Data were saved in hard discs and were converted to DICOM (Digital Imaging and Communications in Medicine) format. Then, the DICOM files were imported to MevisLab (MeVis Medical Solutions

AG, Fraunhofer MEVIS, Bremen & Lübeck, Germany) software for analysis. CBCT images were superimposed using maxillary bone and teeth as landmarks. The buccal area of the implant was isolated and the buccal bone was automatically selected by the software based on upper and lower grey value control thresholds.

The lower grey value control threshold was the highest grey value of the gingiva palatal to the tooth contralateral to the implant site plus one grey value. The upper control grey value threshold was the highest grey value of the cortical bone of the mental area of the lower border of the mandible. The superimposed images were exported as DICOM files to MATLAB software and the initial buccal bone thickness

56 (1st CBCT) and the buccal bone difference between the two CBCTs were calculated for each patient. Then, initial buccal thickness and buccal thickness difference of only the midbuccal area (middle third mesiodistally) of the buccal bone of the implant was selected for data analysis.

Registration of images, software development, selection of study areas and calculation of initial bone thickness and difference in bone thickness between the two scans was done by one medical imaging specialist (M.D.). Selection of control areas was done by a clinician (V.K.). More detailed information about CBCT analysis and related discussion was provided in Chapter II.

Statistical analysis

Sample size was determined by using A-priori calculation method and based on our previous work (Alssum et al 2017). All clinical measurements were done by two trained examiners (VK or BL). A training session was conducted prior to initiation of the current and previous studies to control intra- and inter-examiner differences for clinical parameters. Training related to CBCT analysis was provided in Chapter II. LDF recordings were calculated as the average of the readings from the 2-minute-period of each recording by the Periosoft software program (PSW 2, version 2.5.5, PerimedAB). Changes in blood perfusion were expressed as the percent difference (ΔPU%) between the PU value at a specific site at a specific

57 observation time point (PUt) and the individual baseline value of the same site (PU0) using the following formula:

ΔPU% = PUt - PU0 / PU0 x 100

Descriptive statistics are reported as mean±se and percentage. Patient was chosen as unit of measurement. Data were analyzed in GraghPad Prism 5

(GraphPad Software, Inc. CA, USA) and statistical Analysis Software, version 9.3 (SAS

Institute Inc, Cary, NC, USA). A linear mixed model regression analysis was used for repeated continuous measures fixed and random effects within and between groups. A random effect (intercept and slope) regression analysis was conducted to estimate the slopes of the outcome over continuous time for non-grafted and grafted groups. For repeated measure binary outcomes, generalized estimating equations

(GEEs) was used. T-test, chi-square test or Wilcoxon-Man-Whitney test, as appropriate, was used to analyze all the other non-repeated data. Spearman

Correlation Coefficient was used for correlation analyses. p≤0.05 was chosen as statistically significant value (in order to reject null hypothesis).

Results

Study population

Twenty-seven patients were recruited and 24 completed the study (13 males, 49±4 years). Two subjects were excluded at the time of surgery due to

58 changes in surgical protocol and 1 withdrew from the study after the 1-month- follow-up appointment due to moving out of the country. Demographic details related to study population were provided in Table I. Each subject contributed with a single site resulting in 24 total surgical sites. 9 patients belonged to the non- grafted group and 15 patients belonged to the grafted group. Patients in the non- grafted group never received bone graft at the research site before or during the implant placement surgery. Out of the 15 patients in the grafted group, 8 received an implant after extraction and socket preservation, 4 after guided bone regeneration and 3 had immediate implants with simultaneous bone graft placement. Mean implant diameter and length were 3.9±0.1 mm (3.3-4.8mm) and

11.2±0.2 mm (10-13mm), respectively. The implant systems that were used included Zimmer TSVT, Straumann ROXOLID, Astra OsseoSpeed EV, 3i and Nobel

Biocare Conical Connection. 16% of the implants were placed as 1-stage implants.

Demographics are presented in table 1.

Clinical parameters

Mean initial PD of all subjects was 1.85±0.29 mm with no statistically significant differences between two groups (p=0.35 between groups). Minimal changes in PD were observed following 4 months of healing (data not shown). 58% of the cases (14 out of 24) were initially diagnosed with thin soft tissue biotype with similar distribution for grafted and non-grafted groups (Table 1); The percentage of thin biotype cases was higher in non-grafted group (78% [7 out of 9]) compared to

59 grafted group (44% [7 out of 16]; p<0.05). Mean initial keratinized tissue width was

4.61±0.67 mm in the non-grafted group and 4.21±0.54 mm in the grafted group before the surgery (p=0.66 between groups). 4 months after surgery this measurement was 4.72±0.79 mm and 4.16±0.51 mm for non-grafted and grafted group respectively (p=0.54 between groups; p=0.79 within non grafted group, p=0.52 within grafted group) between and within groups).

Percentage of plaque and gingival bleeding sites specific to surgical sextant are shown in figure 14. At baseline, 30% of sites of the treatment sextant at non- grafted group and 25% of sites at grafted group were plaque positive. In both groups, the number of sites with detected plaque increased at 3 days (non grafted

35% and grafted 27%) and then decreased and reached baseline levels at 6 days

(non grafted 30% and grafted 23%). At 9 days and 1 month, plaque levels remained similar to 6 days in the grafted group (23% and 25% respectively). However, in grafted group, there was an increase in plaque levels from 9 days to 1 month (27% and 35% respectively, p=0.78). As reported, at all time points, there was a 5-10% difference in number of sites with detected plaque between the groups with non grafted group accumulating more plaque, however at 4 months plaque was the same

(17%) and lower than baseline in both groups. The number of sites with detected plaque at 4 months was significantly decreased compared to 3 days and 1 month in non grafted group (p=0.02 and p=0.03 respectively).There was no statistically significant differences between groups at any time point (p>0.05; figure 14). In general, gingival bleeding was observed more often in grafted group compared to

60 non-grafted group throughout the study period (figure 14); At baseline, gingival bleeding occurred at 4% of sites in the non-grafted group and 8% of sites in the grafted group. An increase in gingival bleeding was faster and more persistent in grafted group occurring at 3 days (13%), 6 days (12%) and 1 month (13%). In the non grafted group an increase occurred only at 6 days (9%). At 9 days and 4 months, the number of sites with bleeding was similar to baseline in both groups (non grafted 1% and 3% and, grafted 8% and 6%, respectively). Even though there are fluctuations in number of sites with gingival bleeding within and between groups, none of the differences were statistically significant (p>0.05; Figure 14).

Healing was uneventful in all patients (Figures 15A and 15B). At day 3, wound closure was achieved in 11% and 6% of the sites in the non grafted and grafted group, respectively. None of the wounds were clinically open by 1 month in the non grafted group while 13% of the wounds were still open at 4 months for the grafted group (p<0.01; Figures 15A and 15B). Hydrogen peroxide test was performed as a secondary confirmation for wound exposure. Related data was in agreement with clinical evaluation of wound closure (Figures 15A and 15B).

Similarly, wound area appeared more erythematous in grafted group for a longer period of time (Figures 15A and 15B; p=0.03, between groups). At 9 days, 78% and

100% of the wound areas were erythematous in non-grafted and grafted group, respectively. At 1 month, erythema was evident in 22% and 31% of wound areas in non grafted and grafted sites. Interestibgly, erythema persisted in one subject

61 representing 11% of the wound areas in the non grafted group up to 4 months

(Figures 15A and 15B).

Bleeding at wound site was experienced in 89%, 56%, 33%and 0% of the patients in the non grafted group and, 69%, 56%, 31% and 6% of the patients in the grafted group at 3, 6, 9 days and 1 month (p>0.05; between group differences).

Necrosis was evident in 11% of the patients in the non grafted group until 9 days and, in 19%, 6% and 0% of the patients in the grafted group at 3, 6, and 9 days, respectively (p>0.05; between group differences). No patient at any time experienced graft mobility or suppuration (data not shown).

In both groups, patients experienced minimal pain after surgery (Figure 16A and 16B). At 3 days, pain levels ranged from 0-4 in VAS with a median of 1 (56% of the patients) in non grafted group and a median of 2 (46% of the patients) in grafted group indicating that grafted group experienced more pain than non grafted group

(p<0.01). At 6 and 9 days the majority of patients experienced no pain (78% and

100% of patients in non grafted group and, 68% and 78% of patients in grafted group, respectively).

Changes in Wound Fluid Volume

The amount of gingival crevicular fluid (GCF) and wound fluid (WF) collected during the study period is presented in figure 17. At baseline, GCF levels were similar in both groups (non grafted 0.83±0.15μl and grafted 1.27±0.2μl; p=0.13, between groups). Wound fluid volume increase followed a faster and more

62 persistent response at grafted sites peaking at 3 days (1.66±0.3 μl; p<0.01) and 6 days (1.29±1.9 μl, p=0.04) compared to a 6-day-peak at non grafted sites (1.92±0.3

μl, p<0.01). However, at 6 days WF volume was higher in non-grafted sites compared to grafted sites (p=0.01, between groups). The wound fluid volume reached baseline levels in both groups at 9 days (1.28±0.22 μl and 1.28±0.23 μl in grafted and non-grafted groups, respectively) and remained at baseline levels at 1 and 4 months (1.31±0.2μl and 0.84±0.12μl) for non grafted group and, 0.93±0.1μl and 0.96±0.14μl for grafted group, respectively) (Figure 17).

LDF measurements

Figures 18 through 20 are presenting post-surgical gingival blood perfusion rate. As expected, surgical manipulation required to place dental implant device caused a statistically significant decrease in gingival (flap) blood perfusion representing surgery related trauma and ischemia (Figures 18 and 17; p<0.01 compared to baseline). The immediate effect of surgery on blood perfusion was similar between grafted and non-grafted sites (Figure 19; p>0.05 between groups).

In fact, it was possible to repeat this data by comparing it with our previous work on blood perfusion rate following bone regeneration procedures within similar anatomical locations (Figures 19 and 20). At 3 days following implant placement surgery, the blood perfusion in the non-grafted group was 57% of the baseline blood perfusion and 42% of the baseline blood perfusion in the grafted group (Figure 19).

Despite the fact that these differences were almost 50% of the baseline blood

63 perfusion, they were not statistically significant from baseline (Figure 19, p>0.05).

After 3 days, both groups exhibit an ischemic response until 1 month. At 6 days, 9 days and 1 month, blood perfusion reached 6%, 1% and 6% of baseline blood perfusion in non grafted sites and it reached 17%, -16%, -20% of baseline blood perfusion in grafted sites (Figure 19). At these time points, decrease in blood perfusion was statistically significant compared to baseline in both groups (baseline to 6 days p=0.3, baseline to 9 days p=0.02, baseline to 1 month p=0.03 in non- grafted group and, baseline to 6 days p<0.01, baseline to 9 days p<0.01, baseline to 1 month p<0.01 in grafted group) At 4 months, grafted sites remained ischemic reaching -45% of the baseline blood perfusion (p<0.01). However, non grafted sites recover 54% of the baseline blood perfusion, which is similar to 3-day-recovery

(Figure 19; p>0.05 baseline to 4 months and p>0.05 3 days to 4 months). When comparing to our previously published work on blood perfusion rate following bone regeneration procedures (Alssum et al., 2017), post-implant surgery non-grafted group response following 4 months of healing was similar to post-GBR group response while post-implant surgery grafted group presented continuous ischemic response up to 4 months (Figure 20).

Resonance Frequency Analysis

ISQ data are presented in figure 21. Baseline mean ISQ was 69 for both groups with greater variation observed in non-grafted sites (Figure 9; 69±4 and

69±1, non-grafted and grafted sites, respectively). An increase in mean ISQ values

64 was observed for both groups (72±3 and 74±2, non-grafted and grafted sites, respectively). Time-dependent change was statistically significant only for grafted sites (p<0.01).

CBCT analysis

Initial buccal bone thickness for non-grafted and grafted sites was 1.52±0.05 mm (1.3-1.6 mm) and 1.78±0.1 mm (1.5-2.1 mm), respectively (Figures 22, 23, 24A and 24B). There was no statistically significant difference in initial buccal bone thickness between grafted and non-grafted groups at any coronal-apical vertical levels (Figure 24A and 24B; p>0.05). Following 4 months of healing, CBCT analysis revealed a mean loss of 0.1±0.15 mm ranging from a loss of 1mm to a gain of 0.5mm in non-grafted sites and a mean loss of 0.3±0.24 mm ranging from a loss of 1.5mm to a gain of 1.3mm in grafted sites (Figure 25; p<0.01, between groups). The most pronounced bone thickness loss was recorded at 6-8 mm along the implant length

(Figure 26; p=0.02, within grafted group). At the most coronal 2 mm along the implant length, where the flap was elevated, grafted sites with initial buccal bone thickness ≥1.5 mm lost more bone thickness compared to sites with initial buccal bone thickness <1.5 mm (p<0.01). Among sites with thin tissue biotype at baseline, grafted group lost more bone thickness compared to non-grafted group at 0-6mm along the implant length (p<0.05 between groups). At 4 months, mean buccal bone thickness was 1.52±0.14mm (0.2-4.6 mm) and 1.5±0.11 mm (0.2-4.1mm) in grafted and non-grafted group, respectively (p>0.05).

Correlations

Thick soft tissue biotype was correlated with less buccal bone thickness loss at the apical 10-12mm along the implant length in both groups (r=0.8, p=0.03). Also, implant stability quotient was positively correlated with implant diameter in both groups (r=0.3, p=0.04). In grafted group only, thin tissue biotype was correlated with higher wound fluid production (r=0.4, p=0.03).

Discussion

Short- and long-term post-tooth extraction and peri-implant buccal bone integrity has been investigated through several case series as well as systematic reviews.16, 49 The effect of surgical trauma on buccal bone remodeling is well established.65 However, the recovery rate of buccal flap blood perfusion especially at a grafted site has not been investigated. We previously reported a continuous hyperemic response at sites treated with bone regeneration procedures.41 The purpose of the current study was to determine the rate of recovery from surgical trauma through blood perfusion following implant placement surgery and detect differences between grafted and non grafted sites.

Adult patients receiving a single maxillary non-molar implant were recruited for this prospective observational study. This implant location was selected due to the data of various studies indicating that the buccal bone of the esthetic area

66 undergoes significant resorption after extraction. 8, 9 Non smoking patients 18-75 years old in good systemic and periodontal health were recruited in order to control for effect of age, local and systemic diseases and smoking on wound healing and gingival blood perfusion. Five different implant systems were chosen with a mean diameter of 3.9±0.1 mm (3.3-4.8mm) and a mean length of 11.2±0.2 mm (10-

13mm). Despite the differences in thread design and roughness/porosity of the implant surface, all implants were root form, screw type and placed at bone level.

Related preferences were patient specific and not study directed.

Our results indicate that soft tissue healing in grafted sites appeared to have a higher inflammatory clinical profile as expressed in wound fluid volume, erythema and delayed wound closure compared to non grafted sites. Namely, in grafted sites the wound fluid volume increase was faster and more persistent peaking at 3 days and remaining increased till 6 days compared to non grafted sites where wound volume followed a more gradual increase till 6 days. The recovery rate of the wound fluid volume was similar in both sites reaching baseline GCF levels at 9 days. This increase in wound fluid volume is expected after surgery and was in accordance with previous studies on peri-implant would healing.67 Clinically, the wound areas in grafted group appeared more erythematous for longer period of time compared to non grafted group. In particular, at 9 days, 22% and 78% of the wound areas were erythematous in non-grafted and grafted group, respectively. Furthermore, wound closure over the implant was slower in grafted group compared to non- grafted group. Primary wound closure immediately following surgery was observed

67 only in two cases (8% of the study population; one grafted and one non-grafted sites). At 9 days, 22% of the wounds in the non grafted group appeared closed compared to none in the grafted group. Despite all wound sites in the non grafted group being clinically closed by the 1st month of healing, only 87% of the wound sites in grafted group were clinically closed at 4 months. Failure of wound closure described as early implant exposure following two-stage implant placement surgeries is a well-documented factor of crestal bone loss;68 however its effect on regeneration outcomes following GBR procedures were reported as minimal and its effect on buccal bone thickness has not been investigated. Our current study did not find any correlation between wound closure and final peri-implant buccal bone thickness. Interestingly, our previous work on post-extraction bone regeneration procedures reported an association between the amount of wound exposure mesiodistally and the bone fill outcomes. 41

Buccal flap blood perfusion using LDF was also monitored as a measure of soft tissue healing in our study. In accordance with previous studies,36, 39, 69 the gingival blood perfusion decreased significantly after surgery. This decrease is attributed to the disturbance of blood supply during flap elevation and the use of local anesthetic with vasoconstriction. In previous studies,69 the gingival blood perfusion after local anesthesia alone initially decreased and then reached baseline levels after 65 minutes. In studies where a flap was elevated after local anesthesia administration, the immediate decrease in blood perfusion lasted for at least 2 hours.39 In the present study, surgical procedures lasted for up to 150 minutes

68 confirming previous decreased blood perfusion published data.39, 41 At 3 days, blood perfusion increased in both groups reaching 57% and 42% of the baseline blood perfusion in the non grafted group and grafted group, respectively. An ischemic pattern persisted in both groups up to 1 month post-operatively. Non-grafted sites had a 4-month recovery level comparable to 3 days post-op (p>0.05). However, grafted sites presented with a significant ischemic response even at 4 months post- implant placement surgery (p<0.01). In contrast to our data, previous studies reported a hyperemic response after flap elevation that persisted up to day 7 postoperatively. The blood perfusion rate returned to baseline 15 days after the surgery.39 Furthermore, Alssum et al41 reported that after extraction and socket preservation or guided bone regeneration procedures the flap remained hyperemic until 4 months post-operatively. To the best of our knowledge, this is the first study that reports a continuous ischemic response measured by LDF 4 months after flap procedures. Since 4-month-ischemia is present only at grafted sites (Figure 8), it is speculated that soft tissue manipulations during previous surgeries are responsible for scar tissue formation within the flap. For these sites, the implant placement surgery is the second surgery at the same area, therefore more scar tissue may occupy the area. This is supported by a prospective study by Nakamoto et al reporting that blood perfusion measured using laser speckle imaging was lower in healthy gingiva around maxillary anterior implants compared to pristine healthy gingiva of adjacent teeth.70 The differential response may also be due to regenerated

69 bone tissue in addition to residual graft materials remaining within the regenerated tissue as well as at the soft/hard tissue interface.

Blood perfusion changes were independent of tissue biotype with statistically non significant differences between sites with thick or thin biotype at baseline or 4 months. This may be related to the 1mm3 space limitation of LDF.

Since light from LDF probe does not penetrate the tissues deeper than 1mm71, blood perfusion changes in deeper tissue layers of thick tissue biotype flaps could not be detected, indicating possibly false negative results that there are no differences in blood perfusion between thick and thin tissue biotype flaps.

Mean baseline implant stability quotient was high in both groups. Only one implant in the non grafted group had low baseline ISQ values due to significant surgical maneuvers in order to correct the angulation of the osteotomy. At 4 months,

ISQ values increased in both groups, but the increase in grafted group was statistically significant. Deli et al reported time-dependent results indicating that time of implant placement after bone grafting affects implant stability.46 In their study, ISQ values of implants placed in regenerated bone after 12 months of healing were superior to those of implants placed in pristine or regenerated bone after 6 months of healing.46 Deli et al’s data combined with our data suggest that regenerated bone undergoes changes with possible differential response affecting tissue integrity at the interface between the implant and the bone.

CBCT analysis revealed a mean loss of 0.1±0.15 mm (-1+0.5 mm) and 0.3±0.24 mm (-1.5+1.3 mm) in non-grafted and grafted sites, respectively (Figure 12; p<0.01,

70 between groups). The most pronounced bone thickness loss was recorded at 6-8 mm along the implant length (p=0.02, within grafted group). Thus, despite the surgical trauma, buccal bone loss during early phases of healing is minimal and in agreement with previously reported bone loss due to flap elevation alone.34 Initial bone thickness was a determinant of buccal bone loss at the most coronal 2mm where the flap was elevated only in grafted group. In fact, sites with initial buccal bone thickness ≥1.5 mm lost more bone thickness compared to sites with initial buccal bone thickness <1.5 mm (p<0.01). This is in contrast to the observation by

Wood et al34 that after flap elevation sites with thin bone undergoes more crestal bone loss than sites with thick bone. This difference may be related to the fact that

Wood et al investigated crestal bone loss around teeth with periodontal disease and not buccal bone loss in sites without teeth. Furthermore, since this observation was significant only for grafted sites, bone graft material that is lost after flap elevation may have contributed to the initially increased buccal bone thickness. This clinical study could not further evaluate the effect of residual bone graft particles on remodeling of bone in the cellular level, however this is an area that needs to be further investigated. Another significant outcome that may be related to the residual bone graft material is that the most pronounced bone thickness loss was recorded at

6-8 mm along the implant length (p=0.02, within grafted group only). It is well documented that after extraction and regenerative procedures, the most pronounced buccal bone thickness loss occurs at the most coronal part of the socket during early healing and before implant placement.54 The area that accommodates

71 the second largest quantity of bone graft is the middle part of the root, since the root form is similar to a cone. It may be speculated that residual bone particles that initially seemed integrated in the middle part of the socket can contribute to buccal bone thickness loss after mechanical stress during implant placement. The middle area of the implant experiences more mechanical stress during osteotomy preparation since osteotomy angulation corrections are usually done at this area. At

4 months, buccal bone thickness was 1.52±0.14mm (0.2-4.6 mm) and 1.5±0.11 mm

(0.2-4.1mm) in non-grafted and grafted group, respectively (p>0.05). Even though mean final thickness values are acceptable, as defined by buccal bone thickness ≥

1mm, only 33% (3/9) implants in non-grafted and 45% (5/11) of implants in grafted group had acceptable buccal bone thickness along their entire buccal wall at

4 months. The higher percentage of implants in grafted sites with adequate buccal bone thickness justifies the importance of previous bone grafting.

This study, however, has certain limitations. The sample size indicated by power analysis was not achieved for the non-grafted group (n=9 instead of n=15).

Although the sample size was not ideal, statistically significant differences within and between groups were identified indicating the possible robustness of the differences. Another limitation is the difficulty in controlling various surgical modalities especially for sites with bone grafting indications; out of the 15 patients in the grafted group, 8 received an implant after extraction and socket preservation,

4 after guided bone regeneration and 3 had immediate implants with simultaneous bone graft placement. Buccal bone loss is well documented during all three

72 procedures during early wound healing, however the buccal bone thickness loss after extraction and immediate implant placement is not prevented due to implant placement. Namely, in a randomized controlled clinical trial by Sanz et al,72 4 motnhs after immediate implant placement buccal bone thickness loss was 1.1mm in sites where the gap between the implant and the buccal bone wall received bone graft and 1.6mm where the gap was not grafted. In these studies, the values of buccal bone loss after extraction and immediate implant placement with bone graft in the gap, as done in our study, and the values after extraction and socket preservation in a systematic review by Horvath et al were similar (1.1mm vs

1.12mm).16 Therefore, the buccal bone thickness changes that are reported here may be more indicative of changes after extraction than changes after implant placement. Lastly, another limitation lies in the interpretation of the LDF data. The

PU recordings represent relative changes in the flux of blood cells multiplied by their velocity, therefore they cannot be used as absolute values. In our study, LDF changes were presented as the percentage change from baseline. Even though this

LDF data presentation has been used also by other studies36, 39, 41, it has not been proven if it is the most accurate way to present blood flow changes.

Conclusion

This is the first clinical study to compare both soft and hard tissue early healing outcomes following implant placement surgery in non-grafted and grafted sites.

73 Post-implant placement early wound healing in grafted sites is characterized by a continuous ischemic response and a higher inflammatory clinical healing profile compared to non-grafted sites. However, despite the surgical trauma associated with flap elevation and implant insertion, buccal bone thickness loss is minimal at both grafted and non-grafted sites and consistent with previously reported bone thickness loss after flap elevation alone. Thin tissue biotype in grafted sites may be a contributing factor to greater bone loss. Although grafted sites lost statistically significantly more buccal bone thickness than non-grafted sites, the additional bone loss in grafted sites is not considered clinically significant. However, more grafted sites appear to have adequate final buccal bone thickness at 4 months compared to non-grafted sites. The study results highlight the fact that there are distinct differences in early wound healing between non-grafted and grafted maxillary esthetic zone sites after implant placement. The possible significance of these reported differences on long-term buccal bone integrity, especially at grafted sites, remains to be established.

74 Table 1. Demographics for grafted and non-grafted groups.

Grafted (N=16) Non-grafted (N=9) Age 50±4 yrs 47±7 yrs Gender 10 females 2 females 6 males 7 males Anatomical location 6 incisors 1 incisor 2 canines 2 canines 8 premolars 6 premolars Implant length 11.16±0.27 mm 11.17±0.33 mm Implant diameter 3.95±0.08 mm 3.88±0.1 mm Tissue biotype 7 thin 7 thin 9 thick 2 thick Probing depth (BL) 1.8±0.1 mm 1.96±0.14 mm Keratinized tissue width 4.21±0.54 mm 4.61±0.67 mm (BL)

Figure 14. Plaque and gingival bleeding in treatment sextant; Changes in percentage of sites with plaque was significant only in the non grafted group between 3 days and 4 months (p=0.02) and 1 month and 4 months (p=0.03). Changes in gingival bleeding were not statistically significant in grafted and non- grafted sites.

oups.

Differences in open wound (p<0.01), hydrogen . (B)

grafted sites

and

(A)

grafted sites -

non

peroxide test (p<0.01) and erythema (p<0.01) were statistically significant between the gr

. Wound healing parameters in

igure 15 A F

(B)

grafted sites and

(A)

sites B

grafted - non

Pain Outcomes in

–

VAS

igure 16 F

77 Figure 17. GCF (outlined in purple) and wound fluid (outlined in green) volume. Increase in fluid volume was statistically significant between baseline and 6 days (p<0.01) in non-grafted group and between baseline and 3 days (p<0.04) and baseline and 6 days (p=0.04) in grafted group.

Figure 18. LDF data for the total sample (N=25)

78 Figure 19. LDF data for non-grafted (N=9) and grafted group (N=15); Blood perfusion decrease from baseline during early healing was statistically significant at 6 days (p=0.3), 9 days (p=0.02) and 1 month (p=0.03) in the non-grafted group and at 6 days (p<0.01), 9 days (p<0.01), 1 month (p<0.01) and 4 months (p<0.01) in the grafted group.

Figure 20. LDF data of non-grafted (N=9) and grafted group (N=15) of the current study and post-extraction bone regeneration group (N=15) from Alssum et al study 11

79 Figure 21. Implant stability in non-grafted and grafted group; The increase in implant stability was significant only for the grafted group (p<0.01).

Figure 22. Peri-implant buccal bone thickness for the total sample (N=25) at baseline and at 4 months

80 Figure 23. Peri-implant buccal bone thickness for the total sample (N=25) along the implant length at baseline and at 4 months

Figure 24. Peri-implant buccal bone thickness in non-grafted group (A; N=9) and grafted group (B; N=15) at baseline and 4 months

A B

81 Figure 25. Peri-implant buccal bone loss in non-grafted (N=9) and grafted group (N=15) between baseline and 4 months; The difference between the groups was significant (p<0.01).

Figure 26. Peri-implant buccal bone loss in non-grafted (N=9) and grafted group (N=15) between baseline and 4 months along the implant length; The buccal bone thickness loss was significant only for the grafted group at 6- 8mm along the implant surface (p=0.02).

82 References

1. Atwood DA, Coy WA. Clinical, cephalometric, and densitometric study of reduction of residual ridges. J Prosthet Dent 1971;26:280-295. 2. Benic GI, Hammerle CH. Horizontal bone augmentation by means of guided bone regeneration. Periodontol 2000 2014;66:13-40. 3. Nyman S, Lang NP, Buser D, Bragger U. Bone regeneration adjacent to titanium dental implants using guided tissue regeneration: a report of two cases. Int J Oral Maxillofac Implants 1990;5:9-14. 4. Horvath A, Mardas N, Mezzomo LA, Needleman IG, Donos N. Alveolar ridge preservation. A systematic review. Clin Oral Investig 2013;17:341-363. 5. Ten Heggeler JM, Slot DE, Van der Weijden GA. Effect of socket preservation therapies following tooth extraction in non-molar regions in humans: a systematic review. Clin Oral Implants Res 2011;22:779-788. 6. Van der Weijden F, Dell'Acqua F, Slot DE. Alveolar bone dimensional changes of post-extraction sockets in humans: a systematic review. J Clin Periodontol 2009;36:1048-1058. 7. Valentini P, Abensur D, Densari D, Graziani JN, Hammerle C. Histological evaluation of Bio-Oss in a 2-stage sinus floor elevation and implantation procedure. A human case report. Clin Oral Implants Res 1998;9:59-64. 8. Scarano A, Pecora G, Piattelli M, Piattelli A. Osseointegration in a sinus augmented with bovine porous bone mineral: histological results in an implant retrieved 4 years after insertion. A case report. J Periodontol 2004;75:1161-1166. 9. Degidi M, Piattelli A, Perrotti V, Iezzi G. Histologic and histomorphometric evaluation of an implant retrieved 8 years after insertion in a sinus augmented with anorganic bovine bone and anorganic bovine matrix associated with a cell-binding peptide: a case report. Int J Periodontics Restorative Dent 2012;32:451-457. 10. Retzepi M, Tonetti M, Donos N. Gingival blood flow changes following periodontal access flap surgery using laser Doppler flowmetry. J Clin Periodontol 2007;34:437-443. 11. Alssum L, Eubank TD, Roy S, et al. Gingival Perfusion and Tissue Biomarkers During Early Healing of Postextraction Regenerative Procedures: A Prospective Case Series. J Periodontol 2017;88:1163-1172. 12. Cosyn J, Hooghe N, De Bruyn H. A systematic review on the frequency of advanced recession following single immediate implant treatment. J Clin Periodontol 2012;39:582-589. 13. Chappuis V, Rahman L, Buser R, Janner SFM, Belser UC, Buser D. Effectiveness of Contour Augmentation with Guided Bone Regeneration: 10-Year Results. J Dent Res 2018;97:266-274. 14. Wood DL, Hoag PM, Donnenfeld OW, Rosenfeld LD. Alveolar crest reduction following full and partial thickness flaps. J Periodontol 1972;43:141-144.

83 15. Chappuis V, Engel O, Reyes M, Shahim K, Nolte LP, Buser D. Ridge alterations post-extraction in the esthetic zone: a 3D analysis with CBCT. J Dent Res 2013;92:195S-201S. 16. Sennerby L, Meredith N. Implant stability measurements using resonance frequency analysis: biological and biomechanical aspects and clinical implications. Periodontol 2000 2008;47:51-66. 17. Deli G, Petrone V, De Risi V, Tadic D, Zafiropoulos GG. Longitudinal implant stability measurements based on resonance frequency analysis after placement in healed or regenerated bone. J Oral Implantol 2014;40:438-447. 18. Araujo M, Linder E, Lindhe J. Effect of a xenograft on early bone formation in extraction sockets: an experimental study in dog. Clin Oral Implants Res 2009;20:1-6. 19. Araujo M, Linder E, Wennstrom J, Lindhe J. The influence of Bio-Oss Collagen on healing of an extraction socket: an experimental study in the dog. Int J Periodontics Restorative Dent 2008;28:123-135. 20. Spray JR, Black CG, Morris HF, Ochi S. The influence of bone thickness on facial marginal bone response: stage 1 placement through stage 2 uncovering. Ann Periodontol 2000;5:119-128. 21. Ferrus J, Cecchinato D, Pjetursson EB, Lang NP, Sanz M, Lindhe J. Factors influencing ridge alterations following immediate implant placement into extraction sockets. Clin Oral Implants Res 2010;21:22-29. 22. 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. 23. Huynh-Ba G, Pjetursson BE, Sanz M, et al. Analysis of the socket bone wall dimensions in the upper maxilla in relation to immediate implant placement. Clin Oral Implants Res 2010;21:37-42. 24. Emecen-Huja P, Eubank TD, Shapiro V, Yildiz V, Tatakis DN, Leblebicioglu B. Peri-implant versus periodontal wound healing. J Clin Periodontol 2013;40:816-824. 25. Toljanic JA, Banakis ML, Willes LA, Graham L. Soft tissue exposure of endosseous implants between stage I and stage II surgery as a potential indicator of early crestal bone loss. Int J Oral Maxillofac Implants 1999;14:436-441. 26. 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. J Periodontal Res 2005;40:129-137. 27. 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. 28. Nakamoto T, Kanao M, Kondo Y, et al. Two-dimensional real-time blood flow and temperature of soft tissue around maxillary anterior implants. Implant Dent 2012;21:522-527.

84 29. Kerdvongbundit V, Vongsavan N, Soo-Ampon S, Phankosol P, Hasegawa A. Microcirculation of the healthy human gingiva. Odontology 2002;90:48-51. 30. Aimetti M, Manavella V, Corano L, Ercoli E, Bignardi C, Romano F. Three- dimensional analysis of bone remodeling following ridge augmentation of compromised extraction sockets in periodontitis patients: A randomized controlled study. Clin Oral Implants Res 2018;29:202-214. 31. Sanz M, Lindhe J, Alcaraz J, Sanz-Sanchez I, Cecchinato D. The effect of placing a bone replacement graft in the gap at immediately placed implants: a randomized clinical trial. Clin Oral Implants Res 2017;28:902-910.

85 Chapter IV

Conclusion

The aim of this study was to determine the rate of recovery from surgical trauma through blood perfusion following implant placement surgery. Although the bone quality may be sufficient to place a dental implant, regenerated/regenerating bone may differentially respond to surgical trauma since bone quality, cellular content and angiogenic properties may be different than pristine bone65 41 73. In addition, scar tissue formation due to previous tooth extraction and bone grafting surgeries74 as well as residual graft material left within the regenerated/regenerating bone74 may differentially affect wound healing around the implants placed at grafted sites. Thus, we hypothesized that the recovery rate would be different between previously/simultaneously grafted sites compared to sites that have pristine host native bone. Specifically, we tried to answer the following questions:

86 1. What are the changes in clinical parameters used to evaluate peri-implant early wound healing at anatomically similar grafted and non-grafted sites?

Clinical healing outcomes were uneventful in all cases. However, grafted sites present a higher inflammatory clinical healing profile as expressed by wound closure rate, erythema at the wound site and rate of increase in wound fluid volume.

2. Is the recovery rate of flap blood perfusion following implant placement surgery different between grafted and non-grafted sites?

The results of our study indicate that the buccal flap blood perfusion after implant placement surgery follows an ischemic pattern. Immediately after surgery, the buccal flap blood perfusion decreases and at 3 days postoperatively it reaches approximately 50% of the baseline blood perfusion in both non-grafted and grafted sites. Even though the flap remains ischemic in both sites till 1 month, the flap blood perfusion of non-grafted sites increases till 3-day-blood perfusion levels at 4 months. This does not happen at grafted sites that remain ischemic even at 4 months postoperatively. Although this trend is obvious, the difference in buccal flap blood perfusion levels between sites at 4 months did not reach the level of statistical significance (p=0.06).

87 3. Is the implant stability different between implant fixtures placed into grafted and non-grafted sites during early phases of healing (prior to mechanical loading)?

Baseline mean ISQ was 69 for both groups with greater variation observed in non-grafted sites (69±4 and 69±1, non-grafted and grafted sites, respectively). An increase in mean ISQ values was observed for both groups (72±3 and 74±2, non- grafted and grafted sites, respectively). However, time-dependent change was statistically significant only for grafted sites (p<0.01).

4. Is there any differences in peri-implant buccal bone thickness at grafted and non-grafted sites prior to mechanical loading?

Despite the surgical trauma associated with flap elevation and implant insertion, buccal bone thickness loss is minimal at both grafted and non-grafted sites and consistent with previously reported bone thickness loss after flap elevation alone. Thin tissue biotype in grafted sites may be a contributing factor to greater bone loss. Although grafted sites lost statistically significantly more buccal bone thickness than non-grafted sites, the additional bone loss in grafted sites is not considered clinically significant.

88 Clinical significance and Future Approaches

This is the first clinical study to compare both soft and hard tissue early healing outcomes following implant placement surgery in non-grafted and grafted sites. The study results highlight the fact that there are distinct differences in early wound healing between non-grafted and grafted maxillary esthetic zone sites after implant placement. The findings may be related to tissue changes in grafted sites due to more extensive previous surgical manipulations compared to non-grafted sites. Accumulation of scar tissue and residual bone graft may be responsible for the differences in early wound healing between grafted and non-grafted sites.75, 76 In light of these results, the clinical practice could be adjusted so that less invasive regenerative procedures with newer materials are done. For example, periosteal releasing incisions during bone grafting procedures can be reduced or avoided in order to allow for a more robust blood supply during wound healing and less scar tissue accumulation within the flap. The addition of angiogenic growth factors, such as VEGF, may be of benefit to the blood supply of the area, as seen in flap procedures in the medical literature.77 These, in turn, would allow for provision of more blood supply to the underlying bone. However, the possible significance of these reported differences on long-term buccal bone integrity, especially at grafted sites, remains to be established.

89 References

90 16. Horvath A, Mardas N, Mezzomo LA, Needleman IG, Donos N. Alveolar ridge preservation. A systematic review. Clin Oral Investig 2013;17:341-363. 17. Salvi GE, Bosshardt DD, Lang NP, et al. Temporal sequence of hard and soft tissue healing around titanium dental implants. Periodontol 2000 2015;68:135-152. 18. Schenk RK, Buser D. Osseointegration: a reality. Periodontol 2000 1998;17:22-35. 19. Wetzel AC, Stich H, Caffesse RG. Bone apposition onto oral implants in the sinus area filled with different grafting materials. A histological study in beagle dogs. Clin Oral Implants Res 1995;6:155-163. 20. Valentini P, Abensur D, Densari D, Graziani JN, Hammerle C. Histological evaluation of Bio-Oss in a 2-stage sinus floor elevation and implantation procedure. A human case report. Clin Oral Implants Res 1998;9:59-64. 21. Scarano A, Pecora G, Piattelli M, Piattelli A. Osseointegration in a sinus augmented with bovine porous bone mineral: histological results in an implant retrieved 4 years after insertion. A case report. J Periodontol 2004;75:1161-1166. 22. Degidi M, Piattelli A, Perrotti V, Iezzi G. Histologic and histomorphometric evaluation of an implant retrieved 8 years after insertion in a sinus augmented with anorganic bovine bone and anorganic bovine matrix associated with a cell- binding peptide: a case report. Int J Periodontics Restorative Dent 2012;32:451- 457. 23. Spray JR, Black CG, Morris HF, Ochi S. The influence of bone thickness on facial marginal bone response: stage 1 placement through stage 2 uncovering. Ann Periodontol 2000;5:119-128. 24. Cardaropoli G, Lekholm U, Wennstrom JL. Tissue alterations at implant- supported single-tooth replacements: a 1-year prospective clinical study. Clin Oral Implants Res 2006;17:165-171. 25. Chen ST, Darby IB, Reynolds EC. A prospective clinical study of non-submerged immediate implants: clinical outcomes and esthetic results. Clin Oral Implants Res 2007;18:552-562. 26. Ferrus J, Cecchinato D, Pjetursson EB, Lang NP, Sanz M, Lindhe J. Factors influencing ridge alterations following immediate implant placement into extraction sockets. Clin Oral Implants Res 2010;21:22-29. 27. Lindhe J, Lang NP, Berglundh T, Giannobile WV, Sanz M. Clinical periodontology and implant dentistry. Chichester, West Sussex ; Ames, Iowa: John Wiley and Sons, Inc.; 2015: p. 28. Zweers J, Thomas RZ, Slot DE, Weisgold AS, Van der Weijden FG. Characteristics of periodontal biotype, its dimensions, associations and prevalence: a systematic review. J Clin Periodontol 2014;41:958-971. 29. Chappuis V, Rahman L, Buser R, Janner SFM, Belser UC, Buser D. Effectiveness of Contour Augmentation with Guided Bone Regeneration: 10-Year Results. J Dent Res 2018;97:266-274.

91 30. Cosyn J, Hooghe N, De Bruyn H. A systematic review on the frequency of advanced recession following single immediate implant treatment. J Clin Periodontol 2012;39:582-589. 31. Burkhardt R, Lang NP. Role of flap tension in primary wound closure of mucoperiosteal flaps: a prospective cohort study. Clin Oral Implants Res 2010;21:50-54. 32. Huang LH, Neiva RE, Wang HL. Factors affecting the outcomes of coronally advanced flap root coverage procedure. J Periodontol 2005;76:1729-1734. 33. Fickl S, Kebschull M, Schupbach P, Zuhr O, Schlagenhauf U, Hurzeler MB. Bone loss after full-thickness and partial-thickness flap elevation. J Clin Periodontol 2011;38:157-162. 34. Wood DL, Hoag PM, Donnenfeld OW, Rosenfeld LD. Alveolar crest reduction following full and partial thickness flaps. J Periodontol 1972;43:141-144. 35. Blanco J, Linares A, Perez J, Munoz F. Ridge alterations following flapless immediate implant placement with or without immediate loading. Part II: a histometric study in the Beagle dog. J Clin Periodontol 2011;38:762-770. 36. 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. J Periodontal Res 2005;40:129-137. 37. Baab DA, Oberg A, Lundstrom A. Gingival blood flow and temperature changes in young humans with a history of periodontitis. Arch Oral Biol 1990;35:95-101. 38. Baab DA, Oberg PA. Laser Doppler measurement of gingival blood flow in dogs with increasing and decreasing inflammation. Arch Oral Biol 1987;32:551-555. 39. Retzepi M, Tonetti M, Donos N. Gingival blood flow changes following periodontal access flap surgery using laser Doppler flowmetry. J Clin Periodontol 2007;34:437-443. 40. 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. J Clin Periodontol 2007;34:903-911. 41. Alssum L, Eubank TD, Roy S, et al. Gingival Perfusion and Tissue Biomarkers During Early Healing of Postextraction Regenerative Procedures: A Prospective Case Series. J Periodontol 2017;88:1163-1172. 42. Kokovic V, Krsljak E, Andric M, et al. Correlation of bone vascularity in the posterior mandible and subsequent implant stability: a preliminary study. Implant Dent 2014;23:200-205. 43. Atsumi M, Park SH, Wang HL. Methods used to assess implant stability: current status. Int J Oral Maxillofac Implants 2007;22:743-754. 44. Seong WJ, Conrad HJ, Hinrichs JE. Potential damage to bone-implant interface when measuring initial implant stability. J Periodontol 2009;80:1868-1874. 45. Gehrke SA, da Silva Neto UT, Rossetti PH, Watinaga SE, Giro G, Shibli JA. Stability of implants placed in fresh sockets versus healed alveolar sites: Early findings. Clin Oral Implants Res 2016;27:577-582.

92 46. Deli G, Petrone V, De Risi V, Tadic D, Zafiropoulos GG. Longitudinal implant stability measurements based on resonance frequency analysis after placement in healed or regenerated bone. J Oral Implantol 2014;40:438-447. 47. Hammerle CH, Araujo MG, Simion M, Osteology Consensus G. Evidence-based knowledge on the biology and treatment of extraction sockets. Clin Oral Implants Res 2012;23 Suppl 5:80-82. 48. 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. 49. 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. 50. Araujo M, Linder E, Lindhe J. Effect of a xenograft on early bone formation in extraction sockets: an experimental study in dog. Clin Oral Implants Res 2009;20:1-6. 51. Harris D, Horner K, Grondahl K, et al. E.A.O. guidelines for the use of diagnostic imaging in implant dentistry 2011. A consensus workshop organized by the European Association for Osseointegration at the Medical University of Warsaw. Clin Oral Implants Res 2012;23:1243-1253. 52. Fiorellini JP, Howell TH, Cochran D, et al. Randomized study evaluating recombinant human bone morphogenetic protein-2 for extraction socket augmentation. J Periodontol 2005;76:605-613. 53. 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. J Periodontol 2012;83:581-589. 54. Aimetti M, Manavella V, Corano L, Ercoli E, Bignardi C, Romano F. Three- dimensional analysis of bone remodeling following ridge augmentation of compromised extraction sockets in periodontitis patients: A randomized controlled study. Clin Oral Implants Res 2018;29:202-214. 55. Veltri M, Ekestubbe A, Abrahamsson I, Wennstrom JL. Three-Dimensional buccal bone anatomy and aesthetic outcome of single dental implants replacing maxillary incisors. Clin Oral Implants Res 2016;27:956-963. 56. Schropp L, Wenzel A, Spin-Neto R, Stavropoulos A. Fate of the buccal bone at implants placed early, delayed, or late after tooth extraction analyzed by cone beam CT: 10-year results from a randomized, controlled, clinical study. Clin Oral Implants Res 2015;26:492-500. 57. 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. 58. Leblebicioglu B, Rawal S, Mariotti A. A review of the functional and esthetic requirements for dental implants. J Am Dent Assoc 2007;138:321-329.

93 59. Scarfe WC, Li Z, Aboelmaaty W, Scott SA, Farman AG. Maxillofacial cone beam computed tomography: essence, elements and steps to interpretation. Aust Dent J 2012;57 Suppl 1:46-60. 60. Benic GI, Sancho-Puchades M, Jung RE, Deyhle H, Hammerle CH. In vitro assessment of artifacts induced by titanium dental implants in cone beam computed tomography. Clin Oral Implants Res 2013;24:378-383. 61. Benic GI, Elmasry M, Hammerle CH. Novel digital imaging techniques to assess the outcome in oral rehabilitation with dental implants: a narrative review. Clin Oral Implants Res 2015;26 Suppl 11:86-96. 62. Razavi T, Palmer RM, Davies J, Wilson R, Palmer PJ. Accuracy of measuring the cortical bone thickness adjacent to dental implants using cone beam computed tomography. Clin Oral Implants Res 2010;21:718-725. 63. Naitoh M, Hayashi H, Tsukamoto N, Ariji E. Labial bone assessment surrounding dental implant using cone-beam computed tomography: an in vitro study. Clin Oral Implants Res 2012;23:970-974. 64. Sennerby L, Meredith N. Implant stability measurements using resonance frequency analysis: biological and biomechanical aspects and clinical implications. Periodontol 2000 2008;47:51-66. 65. Araujo M, Linder E, Wennstrom J, Lindhe J. The influence of Bio-Oss Collagen on healing of an extraction socket: an experimental study in the dog. Int J Periodontics Restorative Dent 2008;28:123-135. 66. Kloostra PW, Eber RM, Wang HL, Inglehart MR. Surgical versus non-surgical periodontal treatment: psychosocial factors and treatment outcomes. J Periodontol 2006;77:1253-1260. 67. Emecen-Huja P, Eubank TD, Shapiro V, Yildiz V, Tatakis DN, Leblebicioglu B. Peri- implant versus periodontal wound healing. J Clin Periodontol 2013;40:816-824. 68. Toljanic JA, Banakis ML, Willes LA, Graham L. Soft tissue exposure of endosseous implants between stage I and stage II surgery as a potential indicator of early crestal bone loss. Int J Oral Maxillofac Implants 1999;14:436-441. 69. 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. 70. Nakamoto T, Kanao M, Kondo Y, et al. Two-dimensional real-time blood flow and temperature of soft tissue around maxillary anterior implants. Implant Dent 2012;21:522-527. 71. Kerdvongbundit V, Vongsavan N, Soo-Ampon S, Phankosol P, Hasegawa A. Microcirculation of the healthy human gingiva. Odontology 2002;90:48-51. 72. Sanz M, Lindhe J, Alcaraz J, Sanz-Sanchez I, Cecchinato D. The effect of placing a bone replacement graft in the gap at immediately placed implants: a randomized clinical trial. Clin Oral Implants Res 2017;28:902-910. 73. Iasella JM, Greenwell H, Miller RL, et al. Ridge preservation with freeze-dried bone allograft and a collagen membrane compared to extraction alone for

94 implant site development: a clinical and histologic study in humans. J Periodontol 2003;74:990-999. 74. Klopfleisch R. Macrophage reaction against biomaterials in the mouse model - Phenotypes, functions and markers. Acta Biomater 2016;43:3-13. 75. Miron RJ, Zohdi H, Fujioka-Kobayashi M, Bosshardt DD. Giant cells around bone biomaterials: Osteoclasts or multi-nucleated giant cells? Acta Biomater 2016;46:15-28. 76. Miron RJ, Bosshardt DD. OsteoMacs: Key players around bone biomaterials. Biomaterials 2016;82:1-19. 77. Fang T, Lineaweaver WC, Chen MB, Kisner C, Zhang F. Effects of vascular endothelial growth factor on survival of surgical flaps: a review of experimental studies. J Reconstr Microsurg 2014;30:1-13.