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Bacterial Colonization of the Dental Implant Fixture-Abutment Interface

Permanent Link: http://ufdc.ufl.edu/UFE0042991/00001

Material Information

Title: Bacterial Colonization of the Dental Implant Fixture-Abutment Interface
Physical Description: 1 online resource (76 p.)
Language: english
Creator: TESMER,MICHAEL RAY
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: ABUTMENT -- IMPLANT -- MICROGAP -- MICROLEAKAGE
Dentistry -- Dissertations, Academic -- UF
Genre: Dental Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The geometry of the fixture?abutment interface (FAI) might influence the risk of bacterial invasion of the internal part of the implant. The aim of this study was to use an in vitro model to assess the potential risk for invasion of oral microorganisms into the FAI microgap of dental implants with different characteristics of the connection between the fixture and abutment. Thirty implants were divided into three groups (n = 10 per group) based on their microgap dynamics. Groups 1 and 2 were comprised of fixtures with internal Morse taper connections that connected to standard abutments and the same abutments with a 0.5-mm groove modification, respectively. Group 3 was comprised of implants with a tri-channel internal connection. Fixtures and abutments were assembled and allowed to incubate in a bacterial solution of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) and Porphyromonas gingivalis; chosen based upon the knowledge of their presence in disease of both the periodontal and peri-implant tissues. Two standard abutments were either exposed to bacterial culture or left sterile to serve as positive and negative controls. After disconnection of fixtures and abutments, microbial samples were taken from the threaded portion of the abutment using sterile calcium alginate swabs, plated directly, and allowed to culture under appropriate conditions. Three of the 10 samples in group 1 developed one colony forming unit (CFU) for A. actinomycetemcomitans, whereas zero of 10 samples developed CFUs for P. gingivalis. Ten of 10 and nine of 10 samples from groups 2 and 3, respectively, developed multiple CFUs for A. actinomycetemcomitans and P. gingivalis. This study indicated that differences in implant designs may affect the potential risk for invasion of oral microorganisms into the FAI microgap.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by MICHAEL RAY TESMER.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Koutouzis, Theofilos.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042991:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042991/00001

Material Information

Title: Bacterial Colonization of the Dental Implant Fixture-Abutment Interface
Physical Description: 1 online resource (76 p.)
Language: english
Creator: TESMER,MICHAEL RAY
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: ABUTMENT -- IMPLANT -- MICROGAP -- MICROLEAKAGE
Dentistry -- Dissertations, Academic -- UF
Genre: Dental Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The geometry of the fixture?abutment interface (FAI) might influence the risk of bacterial invasion of the internal part of the implant. The aim of this study was to use an in vitro model to assess the potential risk for invasion of oral microorganisms into the FAI microgap of dental implants with different characteristics of the connection between the fixture and abutment. Thirty implants were divided into three groups (n = 10 per group) based on their microgap dynamics. Groups 1 and 2 were comprised of fixtures with internal Morse taper connections that connected to standard abutments and the same abutments with a 0.5-mm groove modification, respectively. Group 3 was comprised of implants with a tri-channel internal connection. Fixtures and abutments were assembled and allowed to incubate in a bacterial solution of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) and Porphyromonas gingivalis; chosen based upon the knowledge of their presence in disease of both the periodontal and peri-implant tissues. Two standard abutments were either exposed to bacterial culture or left sterile to serve as positive and negative controls. After disconnection of fixtures and abutments, microbial samples were taken from the threaded portion of the abutment using sterile calcium alginate swabs, plated directly, and allowed to culture under appropriate conditions. Three of the 10 samples in group 1 developed one colony forming unit (CFU) for A. actinomycetemcomitans, whereas zero of 10 samples developed CFUs for P. gingivalis. Ten of 10 and nine of 10 samples from groups 2 and 3, respectively, developed multiple CFUs for A. actinomycetemcomitans and P. gingivalis. This study indicated that differences in implant designs may affect the potential risk for invasion of oral microorganisms into the FAI microgap.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by MICHAEL RAY TESMER.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Koutouzis, Theofilos.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042991:00001


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1 BACTERIAL COLONIZATION OF THE DENTAL IMPLANT FIXTURE ABUTMENT INTERFACE By MICHAEL RAY TESMER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGRE E OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011

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2 2011 Michael Ray Tesmer

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3 To my daughter Mary Katherine

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4 ACKNOWLEDGMENTS I woul d like to thank my family, who has supported me throughout all my endeavors without hesitation. Additional ly, I would like to extend my gratitude to the faculty members of the University of Florida department of Periodontology for their contribution to my education and their continuing commitment to our profession.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 6 LIST OF FIGURES ................................ ................................ ................................ .......... 7 ABSTRACT ................................ ................................ ................................ ..................... 8 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 10 2 BACKGROUND ................................ ................................ ................................ ...... 12 History of Osseointegration and Root Form Implants in Restorative Dentistry ....... 12 Evaluation of Dental Implants: Clinical and Radiographic Parameters ................... 16 Evaluation of Osteointegration: ................................ ................................ ........ 19 Evaluation of Peri Implant Mucosa: ................................ ................................ .. 20 Radiographic Evaluation: ................................ ................................ .................. 22 Criteria of Implant Success: Changes over Time ................................ .................... 23 Bacteri al Colonization Patterns of Dental Implants and Peri Implant Infections ...... 25 P. Gingivalis ................................ ................................ ................................ ..... 32 A. Actenomycetemcomitans ................................ ................................ ............. 32 Dental Implant Design and Abutment/Fixture Geometry ................................ ......... 33 Abutment/Fixture Junction Geometry: Laboratory Studies ................................ ...... 37 Abument/Fixture Junction Geometry and the Effects of Placement Position on the Peri Implant Tissues: Animal Studies ................................ ............................ 40 Abument/Fixture Junction Geometry: Human Studies ................................ ............ 42 3 MATERIALS AND METHODS ................................ ................................ ................ 45 Implant Experiment Groups ................................ ................................ .................... 45 Bacterial Culture Condition s ................................ ................................ ................... 46 Microbial Sampling and Detection ................................ ................................ .......... 46 Statistical Analyses ................................ ................................ ................................ 47 4 RESULTS ................................ ................................ ................................ ............... 50 5 DISCUSSION ................................ ................................ ................................ ......... 52 LIST OF REFERENCES ................................ ................................ ............................... 61 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 76

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6 LIST OF TABLES Table page 2 1 Criterion for implant success proposals ................................ .............................. 25 4 1 Median number of colony forming units (interquartile range) for A. actin omycetemcomitans and P. gingivalis by implant group ............................... 51 4 2 Number of implants with a fixture abutment interface microgap contaminated with A. actinomycetemcomitans and P. gingivalis by implant group ................... 51

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7 LIST OF FIGURES Figure page 2 1 Example of the subperiosteal dental i mplant ................................ ...................... 13 2 3 Series of h istolog ic slides after dental implant p lacement ................................ .. 16 2 4 Implant wound chamber filled with a tissue in close contact with the SLA surface representing 4 days of healing ................................ ............................... 17 2 5 Implant wound chamber representing 4 weeks of healing ................................ .. 17 2 6 After 12 weeks the chamber is occupied with mature bone and in cludes also areas of bone marrow in contact with the implant surface ................................ .. 18 2 7 Implants displayed comparing: middle t raditional implant abutment interface rar right p latform switched implant a butment interface ................................ .... 34 2 8 Implants displayed light with dark abutments ................................ ..................... 36 3 1 I mplant of group 1, A butment of group 1, I m plant of group 3, A butment of group 3. ................................ ................................ ................................ .............. 47 3 2 Standard straight abutment of group 2 with 0.5 mm vertical groove. .................. 48 3 3 Implant and a butment of group 1 and group 3 in a plastic container with the bacterial solution. ................................ ................................ ................................ 49 5 1 Internal Morse taper connection with t hreaded solid abutment fixation .............. 52

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8 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science BACTERIAL COLONIZATION OF THE DENTAL IMPLANT FIXTURE ABUTMENT INTERFACE By Michael Ray Tesmer May 2011 Chair: Theofilos Koutouzis Major: Dental Sciences The geometry of the fixture abutment inter face (FAI) might influence the risk of bacterial invasion of the internal part of the implant. The aim of this study wa s to use an in vitro model to assess the potential risk for invasion of oral microorganisms into the FAI microgap of dental im plants with different characteristics of the connection between the fixture and abutment. Thirty implants were divided into three groups (n = 10 per group) based on their microgap dynamics. Groups 1 and 2 were comprised of fixtures with internal Morse taper connections that connected to standard abutments and the same abutments with a 0.5 mm groove modification, respec tively. Group 3 was comprised of implants with a tri channel internal connection. Fixtures and abutments were assembled and allowed to incubate in a b acterial solution of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans ) and Porph yromonas gingivalis ; chosen based upon the knowledge of their presence in disease o f both the periodontal and peri implant tissues. Two standard abutments were e ither exposed to bacterial cul ture or left sterile to serve as positive and negative controls. After disconnection of fixtures and abutments, micr obial sam ples were taken from the threaded portion of the abutment

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9 using sterile calcium alginate swabs plated directly and allowed to culture under appropriate conditions. Three of the 10 samples in group 1 developed one colony forming unit (CFU) for A. actinomycetemcomitans whereas zero of 10 samples developed CFUs for P. gingivalis Ten of 10 and nine of 10 sampl es from groups 2 and 3, respec tively, developed multiple CFUs for A. actinomycetemcomi tans and P. gingivalis This study indicated that differences in implant designs may affect the potential r isk for invasion of oral micro organisms into the FAI microgap.

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10 CHAPTER 1 INTRODUCTION The quantity and quality of the bone surrounding a dental i mplant i nfluences implant osseointegra tion and affects the shape and contour of the ove rlying soft tissues and, conse quently, the esthetic outcome. Only with careful considerations of the biologic principles of peri implant soft and hard tissues, as well as the appropriate selec tion of implant type and position, can a functional and esthetic treatment result be achieved 1 2 Early bacterial coloniza tion around implants by microorganisms associ ated with periodontitis has been re ported 3 5 and this colonizati on of implant surfa ces and peri implant tissues can occur within minutes after implant placement. 6 Whe n a prosthetic abutment is con nected to a fixture, a microgap is created between th e compo nents. Microorgan isms may grow into this fixture abutment interface (FAI) microgap 7 9 and set up a bacterial reservoir, resulting in an area of inflamed so ft tissue facing the fixture abutment junction. 10 A study by Callan et al. 9 used DNA probe analysis to ex amine the bacterial colonization into the FAI in patients. The authors reported modera te to high levels of eight dif ferent putative periodontal pa thogens, includ ing Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans ) and Porphyromonas gingivalis colonizing the FAI. These findings support the results of other researchers 4 5 indicating that normal and pathogenic oral microflora was able to penetrate and colonize the implant abutment interface of dental implants Thus the presence of an FAI microgap in close re lation to bone may have a role in the development of peri implant inflammation and bone loss. 11 16 Furthermore, when using one piece implants that do not have an FAI microgap, minimal early bone re

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11 sorption was found. 12 This result is consistent with the favorable 8 year outc omes of one piece implants in patients reported by Buser et al. 17 The design of the FAI may have an impact on the amount of microbial penetration into the internal part of dental implants. 8 16 18 For instance, in an in vitro study, Quirynen et al. 8 demonstrated the microbial penetration of the FAI m icrogap of fixtures with an ex ternal hex design. However, there was no comparison among implants with different FAI designs in the study. Jansen et al. 18 reported microbial leakage of 13 different implant abutment combinations using Escherichia coli as ind icator bacteria. Among the dif ferent implant abutment combinations, an implant with an internal conne ction and a silicon washer dem onstrated the fewest cases of leakage. In the report by Callan et al., 9 implants from dif ferent manufacturers were used without the authors specifying the characteristics of the FAI ge ometry. Therefore, despite the fact that they reported moder ate to high levels of colonization of the FAI microgap by peri odontal pa thogens, it was not possible to evaluate the impact of the design of the FAI on the microbial penetration. Thus, t here is limited information regarding differences in the mi crobial penetration of the FAI mi crogap of implants with differ ent internal connect ion designs. Hypothesis: It is hypothesized for this study that there is no difference in potential invasion of oral microorganisms into the FAI microgap in dental im plants with different internal connection designs

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12 CHAPTER 2 BACKGROUND History of Osseoin tegration and Root Form Implants in Restorative Dentistry Th e successful replacement of missing natural teeth by tissue integrated root form dental implants has been a major advance in clinical dental treatment. The science behind the osseointegration met hod has evolved over the past several decades in both laboratory and clinical settings. In the early 1950 s bone already had been observed to attach to titanium and was well tolerated as an implant material by various tissues in animal experiments 19 20 Still, in the early 1 960s and 1970 s the idea of the metal implant was far from accepted as a biocompatible material 21 Dr. Per Ingvar Brnemark has been accredited with the early research done in the 1960 s that eventually evolved Although the initial research concept leading to modern day implant osseointegration has been thought of as a chance occurrence. It is without doubt that Brnemark what laid down the foundation for what some have characterized as a shift in paradigm conce rning dental implants 22 Many dif ferent types of implant systems have been used to replace missing teeth, including su bperiosteal implants (Figure 2 1) 23 and blade implants (Figure 2 2) 24 Howeve r, not until 1983 with the widespread introduction of the endosteal osseointegrated dental implant 25 that dental implant s began to gain a wider acc eptance as a replacement for natural teeth and as prosthesis support. In the report on osseointegration of dental implants by Brnemark the indications for treatment we re limited to the edentulous arch 26 In more recent times the osseointegrated dental implant has successfu lly become more widely used in partially edentulous patients, as a single tooth r eplacement 27 29

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13 Fi gure 2 1 Example of the subperiosteal dental i mplant ( s ource: University of Connecticut Health Center http://dentalimplants.uchc.edu/about/types.html l ast accessed January 2011) Fi gure 2 2 Examples e ndosseous osseointegrated dental i mplant s : blade and root f orm. (s ource: University of Connecticut Health Center. http://dentalimplants.uchc.edu/about/types.html l ast accessed January 2011)

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14 The term osseointegration was originally proposed in 1977 in the 10 year report by Brnemark et al. 1977. 26 This concept had been previously outlined in 1969 in an animal study on the experimental use of intra osseous retention of dental prosthesis 30 This term between living bone and the surface of a load carrying i mplant 31 This definition was historically a histologic concept at the level of the light microscope. As this was not a practical or c linically applicable definition; this phenomenon has been defined by several authors from various levels of observation and view points 32 A new definition based on implant stability was suggested by Zarb & Albr e ktsson a s a process whereby clinically asymptomatic rigid fixation of alloplastic materials is achieved and maintained in bone durin g functional loading 33 describe the integration of the fixture into the alveol ar bone 34 The course of osseointegration is achieved through a number of biologic processes, which has been described by Brnemark et al. ( 1969 ) and Schroeder et al. ( 1976 ) 30 34 M ore recently a study by Berglundh et al. evaluat ed osseointegr ation patterns in animals at varying time intervals. 35 A U shaped circumferential trough had been prepared within the thread region of the implants (intraosseous portion), but leaving the tip of each pitch untouched He reby, a secluded area, an experimental wound chamber, was created following implant installation The authors observed that i mmediately after the fixtu re is placed into the osteotomy a blood clot formed and soon became surrounded by inflammatory cells (F igure 2 3) The peripheral portions of the pitches of the thread remain ed in close contact with surrounding bone providing mechanical stability during the initial phase of wound healing. Days later the coagulum is replaced by granulation

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15 tissue containin g the cells needed to form provisional conne ctive tissue. In this newly formed tissue immediately lateral to the titanium surface, the densely packed cells resided in a stroma of fibrin like structures where only a few inflammatory cells are still present (Figure 2 4) As weeks pass an immature woven bone is seen surrounding the implant. This newly formed bone was seen to occupy almost all surface regions of the implant. Bone tissue next to the implant wall was lined with osteoblasts facing a provisiona l matrix rich in vascularity (Figure 2 5) At six weeks large areas of newly formed bone were characterized by the occurrence of primary and secondary osteons, and mineralized tissues were in close contact with the implant surface. After 8 to 12 weeks, n oted signs of remodeling could be seen. Mineralized hard tissue was surrounded by bone marrow, containing adipocytes, vessels, collagen fibers and some mononuclear leukocytes of the mature lamellar bone as the osseointegration process completes at around 3 months (Figure 2 6) 35 O sseointegr ation represents a dynamic pro cess during both its establishment and its maintenance. In the establishment phase, there is a delicate interplay between bone resorption in contact reg ions between the titanium implant body and mineralized bone, Dur ing the maintenance phase, osseointegration is secured through continuous remodeling and ada ptation to function The patterns of bone formati on ob served in the osseointegration model described in the previous study by Berglundh et al. are also consistent with previ ous descriptions of bone model ing and remodeling in bone defects of varying locations and dimensions, including extraction sockets 36 However, it should be realized that the size and

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16 configuration of the w ound defect to undergo bone model ing and remodeling will influence the rate of completio n of the healing process 37 Evaluation of Dental Implants: Clinical and Radiographic Parameters Long term follow up studies are corner stones in clinical evaluations of medical and dental treatment modalities. In the field of dental implants, Adell and co workers presented two classical long term follow up studies 27 38 that have been used to validate t he use of osseointegrated implants to rehabilitate edentulous patients. In their study from 1990 they found that 95% of the maxillae and 99% of the mandibles had continuous prosthesis stability after 15 years. Since then, numerous follow up studies Fi gur e 2 3 Series of h istologic s lides after d ental i mplant p lacement ( a ) I mplant with surrounding soft and hard tissues sampled 2 h after installation g round section o riginal mag x 16 w ound chambers created between th e pitches of the thread ( b ) P itches (arrows) in close contact with the bone tissue w ound chamber filled with coagulum g round section o r iginal mag x 50 ( c ) W ound chamber with coagulum 2 h after de vice installation d ecalcified section o riginal mag x 100 ( d ) C oagulum including large nu mbers of erythrocytes and some in flammatory cells o riginal mag x 400. 35

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17 Fi gure 2 4 I mplant w ound chamber filled with a tissue in close contact with the SLA surface r epresenting 4 days of healing g round section. o r iginal mag x 100 35 Fi gure 2 5 Implant w ound chamber representing 4 weeks of healing o riginal mag. x 1 00 p ortions of the mineralized part of the primary spongiosa are in apparent contact with the SLA surface 35

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18 Fi gure 2 6 After 12 weeks the chamber is occupied with mature bone and includes also areas of bone marrow in contact with the implant surface (ground section; original mag. x 100) 35 on dental implants have been published, and today several studies cover 10 years or more for different patient cat egories 39 46 Although dental implant therapy is regard ed as a safe and reliable procedure, complications do occur. Many authors have discussed different factors that may cause failures in implant treatment, but most likely implant failures have a multi factorial background 47 Esposito et al. 1998 divided implant failures into four groups; biological failures (related to the biological process), mechanical failures of the components (fractures of implants, connecting screws, coatings and prostheses), iatrogenic fai lures ( e.g. nerve damage, wrong alignment of the implant), and functional failures (phonetical, aesthetical, psychological problems). Further, they classified the biological failures as endogenous (systemic and local) and exogenous (operator and biomateri al related). 32 Later, Esposito and co workers ( 1999 ) W hen an implant does not

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19 become osseointegrated it can be regarded as an early failure, in contrast to a late failure resulting from the loss of an achieved osseointegration under functional conditions. 48 Berglundh et a l. described other incidences of biological and technical complications in a (2002) meta analysis. This review included incidences based upon: Implants lost before loading, implants lost during function, persisting sensory disturbance, soft tissue complic ations requiring therapy, peri implantitis, crestal bone loss, implant fracture, complications related to implant components, and complications related to suprastructures. 49 The authors concluded that there is limited information on the incidence of peri implant infections such as peri implantitis as well as the occurrence of crestal bone loss. This information was due to the lack of data describing clinical parameters associated with such incidences. There are several methods used to evaluate dental implants. Some methods mainly focus in evaluating the stability of the dental implant that reflects the status of osseointegration and others focusing on evaluation of the peri implant mucosa that reflects status of health of the soft tissues surrounding the implant. Methods for evaluating the peri im plant tissues include examination and record of: bleeding on probing, suppuration, as well as probing depth measurements relating to marginal bone levels. In addition to those methods, radiographic examination can provide further information regarding the bone topography around a dental impla nt. Evaluation of O steointegration: There are various methods used to evaluate whether osseointegration has taken place or not. A simple method is to test implant stability by exerting a clockwise force on the abutment with an implant driver The implan t can be considered osseointegrated if the implant is found to be immobile 50 Anothe r method is to tap the abutment with a

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20 metallic instrument. A high pitched metallic sound will then indicate an integr ated implant 51 A number of authors 52 54 have reported the potential application of the Periotest in measuring implant mobility. The Periotest device (Siemens, Bensheim, Germany) is an electronic instrument originally designed for quantitative measurements of da mping characteristics of the periodontal ligament to establish a nume rical value for tooth mobility 55 The device is, however, operator sensitive and its value as a clinical diagnostic method to measure implant stability has been questioned 56 58 Resonance frequency analysis (RFA) is another method, developed by Meredith et al. (1996), to evaluate implant stability and found to be of clinical value 59 61 A smal l beam like transducer (Osstell Integration, Diagnostics AB, Partille, Sweden) is attached to the implant or the abutment. The transducer can electronically make the implant to vibrate and the response is measured and registered. The technique is influenced both by the exposed length of the implant and the stiffness of the interface between the implant and the bone. Huwiler et al. (2007) found that even with a good initial implant stability, as measured with the RF A technique, implants might later on fail. 62 Evaluation of P eri I mplant M ucosa: Th e examination of the peri implant tissues around implants has many features in common with th e periodontal examination. The clinical examination must, according to Lang & Lindhe (2003), include parameters such as bleeding on probing, probing depth, and suppuration 63 All these assessments can reveal whether the mucosa around the implant is healthy or not. When probing a pocket around an implant, surrounded by an unhealthy mucosa, the probe goes beyond the sulcus and reaches closer to the bone than it does ar ound a tooth 64 Under healthy conditions the pocket depth, for

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21 conventionally placed implants, ranges b etween 2 4 mm 65 Originally the value of peri implant probing in deter mining the status of the peri implant tissues was questio ned. 66 However, in recent years the usefulness of the information derived from it has generally been accepted 67 Prob ing a pocket is often a difficult task since it is painful for the patient. Further, the assessed pocket depth depends on the pressure applied du ring probing which makes probing operator sensitive and unreliable. Lang and colleagues (2004) stated that peri implant probing should be performed with a light force ( i.e. 0.2 0.25 N) to avoid tissue trauma. 65 Lekholm et al. (1986) found the presence of deep p ockets not to be associated with an accelerated marginal bone loss. 68 Clinical probing depths and radiographic bone level s have been compared to h istological bone level s around screw type implants in monkeys. The radiographic bone level was on average 0.1 0.5 mm, depending on type of implant, short of the histological bone level. The corresponding value for the probing level was much higher, 1.1 3.9 mm. 69 It has been established that bleeding on probing is a valuable parameter in assessing the health status of periodontal tissues 70 71 In particular, the absence of bleeding on prob ing has been shown to be a pre dictor of periodontal stabilit y 72 The size of the tip of the probe as well as the probing force should be standardized to obtain meaningfu l data 73 74 Studies comparing bleeding scores at teeth and implants in the same mouth have reported that the bleeding on probing frequ encies are higher at implants compared to teeth 67 This evidence has been further supported by a (2008) review by Heitz Mayfield et al. where the authors concluded that there is evidence that probing using a light force (0.25N) does not damage the peri implant tissues and that bleeding on probing

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22 indicates presence of inflammation in the peri implant mucosa. The a uthors also stated that probing depth, the presence of bleeding on probing, and suppuration should be assessed regularly for the diagnosis of peri implant diseases. 75 In this context, studies including the health status of the peri implant tissue in their success criteria also assessed the presence or absence of suppuration from th e peri implant sulcus or pocket 17 76 His t ologic studies have shown an inf ltration with large numbers of polymorphonuclear leukocytes in acutely n amed peri implant soft tissues indicating t he clinical diagnostic value of suppuration Radiographic E valuation: Radiography is the most commonly used clinical tool to asses s marginal bone level at implants and its changes over time. The technique of choice is the intra oral radiographic technique The reason is that this permits individual adjustment of the X ray beam angulation relative to each individual fixture. In addition, the high resolution in intra oral radiographs provides possibilities to evaluate the bone level. All radiographs of impla nts should be taken with the film/detector parallel to the implant and the X ray beam directed perpendicular to it. Threaded implants have the advantage of making it easy to determine whether the implant has been depicted with correct vertical irradiation geometry or not. An intra oral radiograph, however, only illustrates clearly the mesial and distal marginal bone levels and early bone loss often occurs on t he facial aspect of the implant. 77 78 Marginal bone loss during the first year of loading has been reported to be at most 1 1.5 mm and thereafter less than 0.2 mm on an annual basis. 38 41 79 80 Little is known a bout the bone loss during the heali ng period. A strand et al. (2004) started to radiographically monitor the marginal bone level at the time of implant insertion and found the bone loss between implant placement and prosthesis insertion to be several

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23 times higher than between prosthesis insertion and a 5 year follow up. 81 Alternatively, m ore recent animal and clinical studies suggest that immediate functional loading of implants with sufficient primary stability may be cons idered a valid treatment alternative in single tooth replacement. 82 It has also been suggested that functional loading of implants may enhance osseointegratio n and does not result in marginal bone loss. 83 However, it has been stated that radiographs are required to evaluate supporting bone levels around implants ; and t hat cone beam radiography offers advantages in implant dentistry that osseous structures can be represented in three planes, true to scale and without overlay or distortion. 75 Criteria of Implant Success: Changes over T ime Over the years many researchers have proposed criteria for success regarding oral implants. One of the oldest, and most commonly used criterion was proposed by Albrek t sson et al. (1986) 50 Albrektsson proposed : Implant immobile when tested clinically ; r adiograph does no t display evidence of peri implant radiolucency ; i mplant has absence of persistent and/or irreversible signs and symptoms such as pain, infections, neuropathies, paresthesia, or violation of the mandibular canal ; v ertical bone loss be less than 1 mm during first year of service ; and a successful rate of 85% at the end of a five year observation period and 80% at the end of a ten year period be a minimum criterion for success 50 This demand for marginal bone loss to be less than 0.2 mm annually after the first year of loading was met w ith much criticism for its rigidity. It has been pointed out that if the bone loss exceeds the yearly 0.2 mm, but then stabilizes and remains equal over a longer period the implant can still be considered clinically successful. 84

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24 a that are similar to those by Albrektsson and co workers (1986) with only minor differences. Albrek t sson & Zarb (1993) suggested that each and every implant should be evaluated as a part of a four grade scale representing success, survival, unaccounted f or and failure. 85 The success category includes implants that meet all of the success criteria according to Albrektsson et al. (1986), and the survival category are those attached implants that are not checked for mobility. The una ccounted for category includes all those patients who died or dropped out of the study, and the failure category includes all removed implants Traditionally implant survival was based upon the ability of the fixture to successfully osseointegrate within the alveolar bone. These requirements for successful osseointegration were introduced by Albrektsson in 1981, and included: biocompatibility, design, surface conditions, status of the host bed, surgical technique at insertion, and loading conditions appl ied afterwards. These requirements for successful osseointegration of the implant are however not necessarily interchangeable with clinical success. Secondary loss of osseointegration may be a frequent problem with respect to different biomaterials as we ll as implant designs. 31 Ongoing marginal bone loss is a factor affecting the outcome of implant treatment. If the marginal bone loss around an implant continues for several years, it may jeopardize the implant outcome 86 If the bone loss is recognized and treated, the implant might be sa ved 87 This topic has been evaluated with regards to implant position, size, and geometry of the implant, resulting in multiple factors with the potential of influencing the bone loss around implants. 88 89 Bacterial colonization of the fixture abutment

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25 interface microgap was one of the factors reported in having an influence on this marginal bone position 18 Table 2 1 Criterion for implant success p roposals Author Crestal Bone Loss Radiography Schnitman & Shulman (1979) Bone loss no greater than a third of the vertical height of the implant No sugges ted criteria Albrek tsson et al. (1986) Vertical bone loss <0.2 mm first year in service No evidence of peri implant radiolucency Smith & Zarb (1989) Albrektsson & Isidor (1993) Wennstro m & Palmer (1999) O st man et al. (2007) Mean vertical bone loss <0.2 mm annually after the first year in service Average bone loss <1.5 mm the first year in service, and thereafter <0.2 mm annually Maximum bone loss of 2 mm between prosthesis installation and the 5th ye ar, with the majority of the loss occurring during the first year Success grade 1 <2 mm bone loss the first year in service Success grade 2 <3 mm bone loss the first year in service No evidence of peri implant radiolucency as assessed on an undistorted radiograph No evidence of peri implant radiolucency No suggested criteria No radiographic signs of pathology No radiographic signs of pathology Bacterial Colonization Patterns of Dental Implants and Peri I mplant Infections The soft tissue surround ing healthy osseointegrated dental implants share anatomical and functional features with the gingiva around teeth. The microstructure has been described i n dog models and human tissues. 90 92 The outer surface of the peri implant mucosa is lined by a stratified keratiniz ed oral epithelium continuous with a junctional epithelium attached to the titanium surface by a basal lamina

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26 hemidesmosomes 93 The 2mm long non keratinized junctional epithelium is in the apical portion only a few cell layers thick, separated from the alveolar bone by 1 2mm of collagen rich connecti ve tissue. This 3 the original mucosal thickness and protects the zone of osseointegration from factors released from plaque and the ora l cavity 94 Unlike the gingiva around teeth, the connective tissue compartment bet ween the junctional epithelium and the alveolar bone consists of a scar like connective tissue almost devoid of vascular structures, greater amounts of collagen and fewer fibrobla sts 91 95 However, more recently the same group examined a 40 wide zone of connective tissue immediately lateral to the implant surface and found that it had many fibroblasts wi th a relatively low proportio n of collagen 96 This may indicate that the fibroblast rich barrier next to the titanium surface has a high cell turnover and that fibr oblasts play an important role in establishing and maintaining the mucosal seal. The inflammatory infiltrate in peri implant tissue and the response to plaque accumulation have been described in animal models 97 98 and hu mans 93 Similar to the disease process o f gingivitis around natural teeth; an inflammatory infiltrate forms in the connective tissue as a response to the microbial colonization of the titanium surface 98 99 The infiltrate represents the local host response to bacterial accumulation and proliferates in an apical direction when the time for plaque accumulation is prolonged 100 The peri implant mucosa is similar to the gingiva around teeth as reg ards of function and immunology 101 An inflammatory cell infiltrate of equal size and composition has been found in clinically healthy tissues of gingiva and peri implant mucosa. 102 Immunohistochemical and immunological analysis show that the inflammatory infiltrate

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27 consists of neutrophils, lymphocytes, macrophages and a few plasma cells. Intraepithelial antigen presenting cells and adhesion molecules, such as ICAM 1 are expressed in epithelia adjacent to implants in a similar fashion as around teeth 103 The distribution of inflammatory cell phenotypes in healthy gi ngiva and peri implant keratinized mucosa is also similar. 104 Functional adaptation of the junctional epithelium occurs although its origin differs from that aroun d the teeth. 105 I n dentate patients with implant sites adjacent to natural teeth, Furst et al. (2007) assessed subgingival plaque samples from implants and neighboring teeth with checkerboard DNA DNA hybridiz ation. Their results concluded that colonization of periodontal bacteria occurred within 30 minutes after the completion of trans mucosal implant installation surg ery. The authors also mentioned that the establishment of the microbiota was faster at toot h sites ; and that different colonization patterns existed at tooth vs. implant sites, suggesting that transmission of pathogens is not immediately established. 6 Bac terial colonization of edentulous patients has been studied in multiple in vestigations 3 106 Implantatio n o f artificial fixtures in patients that are edentulous provides an interesting model for the investigation of shifts in the composition of the microbiota due to alterations of oral ecological conditions. In a study by Mombelli et al. (1988), emphasis was p laced on a limited number of microorganisms known to be associated with various clinical oral conditions Small amounts of bacteria were collected from the preoperative swabs. On an average 86% of the microorganisms were identified morphologically as coc coid cells and over 80% of the cultivated bacteria were Gram positive facultative cocci The authors concluded that a fter implant

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28 install ation, no significant changes in these proportions could be observed Fusobacteria could only be detected in 13 of 10 4 samples. Black pigmented Bacteroides were found in frequently and no trend of increase was apparent in any site over the 180 days of monitoring. 3 Another study looking at microbiol ogical features of implants placed in edentulous patients two years after implantation was investigated by Mombelli et al. (1990). These results indicated that 52% of organisms were facultative anaerobic cocci and 17% were facultative anaerobic rods, whi le Gram negative anaerobic rods accounted for only 7.3%. 106 In edentulous subjects A. actinomycetemcomitans and P. gingivalis are not as frequently associated with peri implant infection as in dentate subjects 107 Danser et al. (1997 ) reported that after full mouth extraction in patients with severe periodontitis, they could no longer detect the latter bacteria on the mucosal surface of edentulous patients, which shows that a shift in the micro flora had occurred after total extraction. A. actinomycetemcomitans or P. gingivalis could not be isolated at the peri implant pockets in these patients after insertion of implants 108 In partly edentulous patients the developing microbiota around implants closely resembles the microflora of natural ly remaining teeth 1 09 A history of periodontitis such as individuals susceptible to periodontal disease and the presence of putative periodontal pathogens are factors that can influence the maintenance and long term prognosis of peri implant tissues in the partly e dentulo us. 75 Quirynen (1996) using phase contrast microscopy, examined partly edentulous subjects and evaluated the impact of periodontitis around remaining teet h and of probing depth around the implants on the composition of the peri implant subgingival flora. 110 They found that the

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29 subgingival microflora around impla nts harbored more spirochetes and motile rods when there were teeth present in the same jaw. The patients were deemed healthy, or as having chronic or refractory periodontitis. Samples fr om deep peri implant pockets proportions of spirochetes and motile rods than those with comparable probing pocket depth in periodontally healthy patients. Papaioannou e t al. (1996) using phase contrast microscopy and DNA probes, determined the prevalence of putative periodontal pathogens in partly edentulous and edentulous patients with a history of periodontal disease. Their microbiological profiles were similar around teeth and dental implants of equal pocket depth, which confirmed the hypothesis that pockets around teeth can act as a reservoir for putative periodontal pathogens. 111 This finding has been confirmed by several clinical studie s of partly edentulous patients 4 109 As early as one month after implantation, putative periodontal pathogens can be detected around the implants of partly edentulous patients 112 Implant failures due to infection are characterized by a complex peri implant microbiota resembl ing that of adult periodontitis. 113 114 Apart from dark pigmented Gram negative anaerobic rods, other bacterial species that associated with peri implant infection include B.forsythus, F.nucleatum, Campylobacter, P.micros and S.intermedi us 115 Other orga nisms not primarily associated with periodontitis, such as Staphylococcus spp, enterics and Candida spp have also been found in peri implant i nfections 116 The longitudinal data available on the microbial colonization of implants in partly edentulous persons with a history of periodontal disease have shown no

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30 association between periodontal pa thogens around teeth and implants with loss of attachment during 36 mo nths function of implants 112 117 Differences in the microbiota for healthy sites as well as peri implantitis has also been investigated. 107 116 118 In 1987 Mombelli et al. found that sites associated with failing implants were characterized by a complex microbiota with a large proportion of Gram negative anaerobic rods. Black pigmen ted Bacteroides and Fusobacter ium spp. were regularly found. Spirochetes, fusiform bacteria as well as motile an d curved rods were a common feature in the darkfield microscopic specimens of these sites. Healthy sites in the same patients harbored small a mounts of bacteria. The predominant morphotype was coccoid cells. Spirochetes were not present, fusiform bacteria, motile and curved rods were found infrequently and in low numbers. 107 Another stu dy further supporting the differences in microbiota between healthy and diseased peri implant tissues was investigated by Leonhardt et al. in (1999). The two types of clinical conditions showed distinct bacterial profiles. For implants with peri implantitis putative periodontal pathogens, such as Porphyromonas gi ngivalis, Prevotella intermedia Prevotella nigrescens an d Actinobacillus actinomycetemcomitans were found in 60% of the cases and microorganisms primarily not associated with periodontitis, such as Staphylococcus spp., enterics and Candida spp., were found in 55% of the peri imaplant lesions. In contrast, imp lants surrounded by healthy tissue demonstrated a microbiota associated with periodontal health. The results indicate that the microbiota of the healthy peri implant sulci is similar to that from corresponding conditions around teeth. However, in peri imp lant areas staphylococci, enterics and yeasts were found

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31 almost as frequently as periopathogens indicating differences as compared to the microbiota around periodontitis affected teeth. 116 To ensure maintenance and long term stability of osseointegrated dental implants, it is essential to study the relation between microbial provocation and th e inflammatory reaction. The inflammation caused by the microbiota probably varies between subjects, as sh own in patients with different types of periodontal disease. Individuals positive for the gene encoding for interleukin times more IL 119 y implants 120 Therefore, the same bacterial stimuli may cause greater tissue destruction in persons with an aberrant host response. Peri implantitis is defined as an inflammatory reaction wi th loss of supporting bone in the tissues surrounding a funct ioning implant 121 specific infection yielding many features in common with or nflammatory, bacterial driven destruction of the implant 107 122 A cause related effect between plaque accu mulation and peri implant mucositis has be en shown in animals and humans 98 99 Moreover the microbial coloniz ation of implants follows the same patter n as around teeth 112 During peri implant breakdown a complex microbiota is established closely resembling th a t found in adult periodontitis 107 113 114 Implants displaying deeper probing depths associated with bone loss and bleeding on probing have been found to harbor A. actinomycetemcomitans and P. gingivalis 114 123 It was partly due to these findings and the virulence factors associated with their pathogenesis that these bacteria were chosen for our investigation.

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32 P. Gingivalis Porphyromonas gingivalis is a Gram negative, non motile, rod shaped, anaerobic organism. To function, it undergoes a mechanism in which it binds to and inhabits the subgingival architecture of the mouth usi ng fimbriae. P. gingivalis is c onsidered an important member of the microb iota involved in periodontal disease progression and bone and tissue destruction 124 This organism is present in very low levels during periodontal health, while during the disease progression of periodontitis can reach significant numbers 125 Putative periodontal pathogens such as P. gingivalis have been found colonizing dental implants within 30 minutes of surgical installation. 6 Leonhardt et al. (1999) provided evidence of implants with peri implantitis, associated with periodontal pathogens, such as Porphyromonas gingivalis 116 P. gingivalis has the unique ability to invade epithelial cells and therefore providing a mechanism by which to escape the protective innate immune response 126 P. gingivalis also produces many cell components and macromolecules that function as virulence factors. Noted virulence factors include t he lipopolysaccharide, proteinases, and collagenases 126 This bacterium is a highly pathogenic and virulent member of the subgingival plaque biofilm. A. Actenomycete mcomitans Aggregatibacter actinomycetemcomitans is a facultative gram negative bacterium, which has been associated with severe oral and non oral infections. A. actinomycetemcomitans has also been associated with peri implant diseases as evidenced by Leonhardt et al. (1999) and has also been connected with breakdown resembling that of periodontitis lesions around implants (peri implantitis). 6 116 A. actinomycetemcomitans has been shown to possess a variety of virulence factors that not only enhance its survival in the oral cavity but also contribute to the pathogenesis of

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33 peri odontitis 127 A. actinomycetemcomitans ability to attach to extracellular matrix proteins and epithelial cells is due to the benefit of adhesins and invasins 128 Once the organism is established in the oral tissues, the host reacts to the bacterial insult with an inflammatory response resulting in the destruction of periodontal tissues in a susceptible host 129 A. actinomycetemcomitans contains a number of factors causing bone resorption including lipopolysaccharide, proteolysis sensitive factor, and GroEL 127 These factors are compounded with its other effects on the connecti ve tissue and extracellular matrix such as collagenase and fibroblast cytotoxin 128 Dental Implant Design and Abutment/Fixture Geometry Altho ugh the basic form of the endosseous dental implant has remained relatively unchanged, the material composition, surface modifications, and abutment connection designs have since changed dramatically There are two basic concepts in implant design; implan ts that are manufact ured to be placed at the tissue level, or at the bone level. The rational behind a tissue level implant is that this concept raises the fixture abutment interface to a supragingival level possibly alleviating the effects of a microgap between the fixture and abutment however this design may have esthetic implications by not allowing for optimal restorative contours or emergence profile. The bone level implant design allows for a more apical connection of the fixture abutment interface This apical connection provides a more ideal emergence profile for the restoration, creating a more natural and esthetic prosthesis. Bone level implant designs have however been shown to be associated with crestal bone loss after installation. 130 131 The original i mplants designed by Brnemark were created with commercially pure titanium using an external hex abutment connection and incorporated a machined external surface. The external hex refers to the implant abutment interface, where the

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34 implant displays a hexagonal connection for which the abutment will be seated (Figure 2 7) This design originally was present to help screw the implant into place The external hex design allow s for an abutment con nection outside of the implant This connection not only provided indexing of single unit abutments but also enabled multiple implants to be rigidly splin ted together via a metal bar for a fixed prosthesis and allow passive connection to the implants To date this implant has supporting documentation spanning over three decades 26 30 132 Although thi s implant was the premier fixture of its time, structural quality and have created a paradig m shift in implant design where recent attention has focused on the fixture surface and the implant abutment interface. The external hex has been replaced in many fixtures with an internal hex design. This internal connection refers to the hexagonal int erface now being inside the implant where the abutment is secured This connection was designed to provide a more ridged connection, enhance implant strength, improve the seal of the fixture and abutment interface, and to medialize this connection. Fi gure 2 7 Implants displayed light with dark abutments A) E xternal hex implant abutment connection with screw fixation B) Internal Morse taper connection with threaded solid abutment fixation C) Internal implant abutment connection with solid threade d abutment fixation, D) Internal Morse taper connection with a press fit fixation.

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35 Another concept that must be realized in understanding implant design is one versus two stage implant surgery. One stage implant surgery refers to placement of a healing ab utment following implant installation that remains transmucosal and exposed to the oral cavity following replacement of the mucoperiosteal flaps In contrast, during two stage implant surgery a cover screw is placed following implant installation and the implant is completely submerged following suturing of the flaps. Three to six months later the implant is uncovered with a second surgical procedure and a healing abutment is placed allowing the peri implant mucosa to heal. Although the one stage techniqu e has less morbidity for the patient, since it involves a single surgical procedure, the two stage surgery might offer greater potential for soft tissue management. This concept of platform switchin g was developed to con trol bone loss after implant placemen t and refers to the use of an abutment of smaller diameter connected to a n imp lant neck of larger diameter 89 connection shifts the perimeter of the implant abutment junction i nwards towards the middle of the implant im proving the distribution of forces while medializing this junction The inward movement of the implant abutment interface is believed to shift the inflammatory cell infiltrate to the central axis of the implant and away from the adjacent crestal bone 89 Degidi et al. 2007, explained that when the horizontal relationship between the outer edge of the implant and a smaller altered, a reduction to crestal bone loss occurs 133 Other authors have supported these findings as well, proving evidence for decreased crestal bone loss following a platform switching concept. 134 136 The ability to reduce or eliminate crestal bone loss would be a major achievement in implant dentistry.

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36 Fi gure 2 8 Implan ts displayed comparing: Midd le Traditional implan t abutment interface Far Right Platform switched implant abutment interface In a two piece implant system the abutment is retained in the fixture using mechanical attachment. This attachment can sometimes result in gaps between t he implant and abutment resulting in a bacterial reservoir. 10 Bacteria in this connection gap may cause an inflammatory process in the peri implant tissue s lead ing to peri implant disease and possible marginal bone loss The introduction of the interna l hex implant gave way for the M orse tapered con nection This internal Morse taper design that so me manufactures have introduced, essentially seals the abutment to the fixture with t his tapered connection. The principle of the Morse taper is that of a cone within a cone, where the male portion and the female portion are both uniformly tapered creating intimate contact Implant taper compresses the walls of the abu tment as it expands. Thus, the stresses inside the materials keep both components fixed together Intimate metal to metal contact of a true Morse taper, may prevent micromovement and transfer the biological width area to a horizontal rather than vertical direction allowing for hard tissue formation above the fixture abutment interface. 137 Different internal connection s

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37 have been evaluated in a recent study where the M orse tapered designs were the only implants failing to show any movement between the abutment and fixture. The results of this study suggested this lack of movement might prevent the pumping of bacterial contaminated liquid from in and out of the im plant, resulting in a decrease of marginal bone loss. 138 The evidence of this Morse tapered design is depicted in our test group. Abu tment/Fixture Junction Geometry : Laboratory Studies In vitro studies evaluating microbial penetration along the internal part of dental implants have been reported utilizing implants with different fixture abutment interface geometries under loading and non loading conditions. In addition, there are laboratory tes ts utilizing finite element analysis trying to demonstrate and evaluate stress patterns on peri implant bone as well as studies looking into the movement of different fixture abutment components. Under non loading conditions, Quirynen et al. ( 1994 ) demon strated that when fixtures with an external hex design and abutments were assembled and installed in a liquid blood medium inoculated with oral microorganisms, bacterial invasion of the fixture abutment interface microgap was detected. 8 Similarly, Jansen et al. ( 1997 ) reported microbial leakage of thirteen different implant abutment combinations using E.coli as indicator bacteria. Among the different implant abutment combinations an implant with an internal connection and a silicon washer demonstrated the fewest cases of leakage. 18 Aloise et al. (2010) compared the frequency of bacterial leakage of Streptococcus sanguinis along the implant abutment interface between two systems of Morse tape r dental implants under non loading conditions Different methods of activation of the taper abutments were used: tapped in and screwed in. Irrespective of which of the two Morse taper implant connection systems of activation was analyzed,

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38 this in vitro experiment showed bacterial leakage along the implant abutment interface. 139 There is limited information from in vitro studies evaluating microbial contamination of the fixture abutment interface microgap under loading c onditions A study by Steinebrunner et al. (2005) evaluated microbial leakage between implants and their abutments using a loading protocol. However, although bacterial leakage along the interface was shown for all tested implants, the number of load cyc les until bacterial penetration occurred differed significantly between implant systems and their connection designs. Specifically, implants with a tri channel internal connection showed bacterial leakage at significantly higher numbers of chewing cycles compared to implants with external hex, implants with internal connection and a silicon washer, and implants with internal hex with friction fit connection 16 Koutouzis et al. ( 2010 ) utilized an in vitro dynamic loading model to assess the potential risk for invasion of oral micro organisms into the fixture abutment interface microgap of dental implants with different fixture abutment connection characteristics. In this experiment tw enty eight implants were divided into two groups (n=14/group) based on their microgap dynamics. Group 1 was comprised of fixtures with internal Morse taper connection that connected to standard abutments. Group 2 was comprised of implants with a four gro ove conical internal connection that connected t o multi base abutments The specimens were immersed in a bacterial solution of Escherichia coli and loaded with 500,000 cycles of 15 N in a wear simulator. Following disconnection of fixtures and abutments, microbial samples were taken from the threaded portion of the abutment, plated and cultured under appropriate conditions. The difference between loosening and tightening torque

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39 value was also measured. One of the 14 samples in Group 1 and 12/14 of sampl es in Group 2 developed multiple colony forming units (CFU) for E.coli. Implants in Group 1 exhibited an increase in torque value in contrast to implants in Group 2 that exhibited a decrease. This study indicated that differences in implant design may aff ect the potential risk for invasion of oral micro organisms into the fixture abutment interface microgap under dynamic loading conditions. Maeda et al. utilized a 3D finite element model to exa mine the biomechanic al advantages of platform swit ching. He not es that this p rocedure shifts the stress con centration away from the bone implant interface, but these forces are then increased in the ab utment or the abutment screw 140 Crestal bone chang es around implants are significantly influenced by microm oveme nt s between the abutment and the implant as shown in animal studies 13 M icromovements of the fixture abutment interface have been evaluated recently with an in vi tro study by Zipprich et al. (2007). Implants from different manufacturers with their respective abutments were loaded up to 200N at an angle of 30 degrees and filmed during force application with a high speed camera. Micromovement creating microgaps wer e recorded in all but 2 of the 9 implant systems one of which was the Morse taper implant described in our study 138 Thus, it can be deduced that an implant with an internal Morse taper connect ion devoid of a microgap created by micromovement between the abutment and the implant may minimize the effect of bacterial infiltrate on the peri implant tissues

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40 Abument/Fixture Junction Geometry and the Effects of Placement Posit ion on the Peri I mpl ant T issues : Animal Studies The location of the fixture abutment interface can be placed in various positions in relation to the alveolar crest (crestal, supracrestal, subcrest al) The location of the fixture abutment interface can be of major importance when the creating esthetic restorations. Placement of the fixture abutment interface in a more apical position can create an ideal emergence profile for the prosthetic construction 141 Subcrestal position of the fixture abutment interface has been reported to have a negative influence on marginal bone level changes in a few ani mal studies. 130 131 142 143 In an experimental study in dogs Hermann et al. ( 2000 ) reported that placement of two part implants with the fixture abutment interface 1 mm below the crestal bone resulted in pronounced crestal bone loss following 6 months of healing. In this study the authors used custom made implants with a fixture abutment interface micro gap of 50 m. 130 Similarly, Jung et al. (2008) evaluated t he influence of non matching implant and abutment diameters on radiographic crestal bone levels in dogs Radiographic analysis rev ealed very little bone loss and a slight increase in bone level for implants placed at the level of the crest or 1 mm above. The greatest bone loss occurred at implants placed 1 mm below the bone crest. No clinically significant differences regarding mar ginal bone loss and the level of the bone to implant contact were detected between implants with a submucosal or a transmucosal healing. However, the amount of crestal bone loss was smaller compared to that found in the study by Hermann et al. ( 2000 ). 131 In a similar animal experiment, Todescan et al. ( 2002 ) evaluated the healing around implants (Brnemark System) that were placed either 1 mm above, level with or 1 mm below the crestal bone.

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41 implant contact was located between 1.6 mm and 2.5 mm apical to the fixture abutment interface with the shortest implant contact distance associated with implants that were placed in the subcrestal posit ion. 143 Similar findings have been reported by Pontes et al. ( 2008 ) where they placed implants with the fixture abutment interface at the bone crest, 1 mm and 2 mm apical to this position. Following 4 months of healing all implant groups had the first bone to implant contact apical to the fixture abutment interface 142 None of these animal studies reported bone formation above the fixture abutment interface when implants are placed in a subcrestal position. In contrast to the previously described studies, few animal experimen ts have reported favorable outcomes for im plants in a subcrestal position with bone formation close to or even above the fixture abutment interface 144 145 Welander et al ( 2009 ) observed osseointegration coronal to the fixture abutment interface when placing implants with the fixture abutment interface 2 mm subcrestally. The test implants in this study had a surface modification extending to the impla nt margin that included the shoulder part of the implant and a conical interface between the abutment and the implant. 144 Similar findings were reported by Weng et al. ( 2008 ) showing that implants wit h subcrestal position presented bone growth onto the implant shoulder in nearly all histological sections. Implants utilized in this study contained a reduced abutment diameter in relation t o the fixture diameter, a Morse taper implant abutment connection, and a microstructured surface treatment which included the cervical collar and extended onto the implant shoulder. 145 Understanding implant placement at different bone level heights and its eff ects on mar ginal bone loss becomes imperative when selecting an implant design. Even though one stage transmucosal implants exhibit stable peri implant bone levels when

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42 the fixture abutment interface is located supracrestal and the border between rough and smooth su rface is located at the alveolar crest it seems that placement of the border between the rough and smooth surface below the bone crest can lead to marginal bone loss and it is not recommended. When placing implants at a subcrestal position, an internal co nnection with a reduced diameter abutment and a Morse taper design may have positive effects on marginal bone levels compared to other designs. Ab ument/Fixture Junction Geometry : Human Studies The effects of the fixture abutment interface have been evalu ated in few clinical studies. 89 133 146 148 The numb er of studies that benefit from histology are further reduced. 133 146 Histologic and radiographic observa tions suggest however that a biologic dimension of hard and soft tissues exists around dental implants and extends apically from the implant abutment interface. This clinical evidence however is beneficial especially when focusing on the comparisons of di ffering fixture abutment interfaces in relation to marginal bone loss 89 In vivo studies evaluatin g histology of peri implant tissues have been reported in the literature. 133 146 Romanos et al. (2005) ev aluated b iopsies fr om human implants This histolog ic and histomorphometric analy sis on the interface of immediately loaded implants retrieved showed a high percentage of bone to implant contacts after a loading period of 2 and 10 months. This observation was independent of the implant system and fixture abutment interface used. The examined implants had a screw geometry and rough surfaces to promote new bone formation at the initial stages of healing dur ing loading 146 Degidi et al. (2008) explained that when the horizontal relationship between the outer edge of the implant and a smaller is altered, a reduction to crestal bone loss occurs After histomorphometr ic analysis of

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43 three Morse taper connection implants the author concludes that when there is zero microgap and no micromovement, platform switching shows no resorption and better esthetics. 133 Lazzara et al. (2006) retrospectively discovered that matching diameter prosthetic components were not available, and many of the early 5.0 and 6.0 mm wide implants received "standard" diameter (4.1 mm) healing abutments an d were restored with "standard" diameter (4.1 mm) prosthetic components. Long term radiographic follow up of these "platform switched" restored wide diameter dental implants has demonstrated a smaller than expected vertical change in the crestal bone heig ht around these implants than is typically observed around implants restored conventionally with prosthetic components of matching diameters. This radiographic observation suggests that the resulting post restorative biologic process resulting in the loss of crestal bone height is altered when the outer edge of the implant abutment interface is horizontally repositioned inwardly and away from the outer edge of the implant platform. This article introduces the concept of platform switching and provides a f oundation for future development of the biologic understanding of the observed radiographic findings. 89 C urrent clinical studies utilizing two piece implant systems with an altered horizontal relationship between the fixture diameter and the abutment diameter, report minimal marginal peri implant bone loss 147 149 In a 5 year prospective study Wennstrm et al. ( 2005 ) reported mean bone level changes from the time of crown placement to the first year follow up of 0.02 mm measured on implant level. 148 Norton et al. ( 2006 ) reported an average of marginal bone loss of 0.65 mm from implant therapy in 54 patients where the implants had been in function for 37 months. 149

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44 Taken together the results of in vitro studies show that differences in implant design may affect the potential risk for invasion of oral micro organisms in to the fixture abutment interface under non loading and dynamic loading conditions. Implants with internal Morse taper connection have the highest potential to prevent bacterial contamination of the fixture abutment interface The results from animal stu dies demonstrate that implants with reduced abutment diameter in relation t o the fixture diameter, a Morse taper implant abutment connection and a microstructured surface treatment which included the cervical collar and extended onto the implant shoulder c an maintain stable peri implant bone levels even when the fixture abutment interface is placed in a subcrestal position. These results are in line with clinical studies showing that implants with reduced abutment diameter in relation to t he fixture diamet er and a Morse taper implant abutment connection exhibit less marginal bone loss compared to implants with an external hex connection at least at the earlier stages of healing With these conclusions some of which were reported after the experimental con clusion of my project, is was the aim of my study to use an in vitro model to assess the potential risk for invasion of oral microorganisms into the fixture abutment interface microgap in dental im plants with different internal connection designs. With th is knowledge it is possib le to reinforce previous conclusions that differences in im plant design may affect t he potential risk for coloniza tion of oral microorganisms into the fixture abutment interface microgap which may ultimately influence the peri imp lant tissues eg. marginal bone

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45 CHAPTER 3 MATERIALS AND METHOD S Implant Experiment Groups For this study, three groups of implants were compared based on their FAI microgap geome try. Ten implants were tested in ea ch experimental group: group 1: fixtures wi th an internal Morse taper connection were connected to standard straight abutments with a hei ght of 6 mm (Fig. 3 1); the abut ments were connected to the fixtures with a torque of 25 Ncm using the appropriate torque wrench according to the man rotocol; group 2: identical fixtures and abutments as described in group 1 were used with the exception that prior to fixture abutment connect ion, a vertical groove of; 0.5 mm depth was prepared with a fissure bur on one side of the abutment (Fig. 3 2). Th e fixtures and abutments were connected using a torque of 25 Ncm using the appropriate torque wrench according to the manu The intro duction of a 0.5 mm groove to the abutment was to ensure microbial penetration to the internal part of the implant, while allowing for the exact same torque for connecting the abutment as the implants in group 1; group 3: fixtures w ith a tri channel internal connection were connected to 3 mm high abutments The compon ents were connected with a tor que of 35 Ncm using the appropriate torque wrench acc ording to the mendation. To evaluate the microbial detection techniques, two standard straig ht abutments with a height of 6 mm were used. These abutments correlated to one negative and one po sitive control abutment The negative control abutment was not connected to a fixture and was not subjected to bacterial culture to ensure an uncontaminated laboratory environment and avoid false positive results The positive control abutment

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46 was not con nected to a fixture but was subjected to the same multi species bacterial culture containing Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis as groups 1 through 3 in order to assure cultivatable bacteria were in fact present in the brot h and that our swabbing and culturing techniques were reliable All fixtures and abutments were connected in a sterile environme nt and placed in a plastic con tai ner with the multi species bacterial s olution covering the FAI inter face and containing microor ganisms as described below Bacterial Culture Conditions Aggregatibacter actinomycetemcomitans VT1169 (State University of New York [SUNY] 465 nalidixic acid resistant rifam picin resistant) was grown in liquid tryptic soy broth supplemented with yeast ex tract and cultured at 37C in 10% CO 2 to the mid logarithmic phase. Porphyromonas gingivalis W83 was grown i n liquid tryptic soy broth sup plemented with hemin, vitamin K, yeast extract, and L cysteine hydrochloride at 37C under anaerobic conditions to the mid logarithmic phase. Implants were placed in an aliquot of a 1:10 dilution of a 1:1 stock solution of A. actinomyce te mcomitans VT1169 and P. gingivalis W83 multi species broth and incubated at 37C under anaerobic conditions for 5 days. Microbial Sampli ng and Detection After disconnection of fixtures and abutments under sterile conditions, microbial samples were taken from the threaded portion of the abutment using sterile calcium alginate swabs. Sample s were plated with the calcium alginate swabs direc tly onto tryptic soy broth agar plates supplemented with yeast extract for the detection of A. actinomycetemcomitans and onto tryptic soy broth agar plates supplemented with hemin, vitamin K, yeast extract, and L cysteine hydrochloride for detection of P.

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47 gingivalis colony forming units (CFUs). Plates A. actinomycetemcomitans and P. gingivalis for were in cubated at 37C in 10% CO2 for 2 and 7 days, respectively. Individual CFUs were counted and recorded. Statistical Analyses Median values and i nterquartile ranges were calcu lated for the number of CFUs for A. actinomycetemcomitans and P. gingivalis In addition, the total number of implants per group exhibiting bacterial coloniza tion of the FAI microgap was calculated. The K ruskal Wallis test with tiple comparisons was applied to evaluate differe nces among the three groups re garding the number of CFUs for A. actinomycetemcomitans and P. gingivalis The X 2 test was used to evaluate differences in the number of implants exhibiting bacterial colonizati on of the FAI microgap among the different groups. A P valu e <0.05 was con sidered significant. Fi gure 3 1. A) Implant of group 1 B) Abutment of group 1 C) Implant of group 3, D) Abutment of group 3.

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48 Fi gure 3 2. Standard straig ht abutment of group 2 with 0.5 mm vertical groove.

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49 Fi gure 3 3. Implant and abutment of group 1 150 and group 3 (left) in a plastic container with the bacterial solution.

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50 CHAPTER 4 RESULTS To validate the colonization and detection techniques, abutments similar to t hose in group 1 and group 3 were left unas sembled and either exposed to bacterial culture o r left sterile acting as positive and negative controls Zero CFUs of A. actinomycetemcomitans or P gingivalis were detected from sampling of abutments that were left sterile (negative control), whereas 188 CFUs of A. actinomycetemcomitans and 113 CFUs o f P gingivalis were detected in samples from abut ments exposed to bacterial culture (positive control). These data indicate th at the conditions for coloniza tion and sample collection were appropriate for the experimental design. To semi quantitate t he a bility of A. actinomycetemcomitans or P gingivalis to colonize the fixture abutment interface micro gap, CFUs from cultured samplings were quantified (Table 4 1). Group 1 exhibited significantly l ower num bers of CFUs for A actinomycetemcomitans (median: 0; interquartile range: 0 to 1) compared to group 2 (median: 81; interquartil e range: 44.5 to 96.5) (differ ence: 36.25; P <0.05) and group 3 (median: 24.5; interquartile range: 11 to 56.5) (difference: 22; P <0.05). There was a significant difference in the number of CFUs for P. gingivalis between group 1 (median: 0; interquartile range: 0 to 0) and group 2 (median: 55; interquart ile range: 35.5 to 96) (differ ence: 35.8; P <0.05). However, the difference in the number of CFUs for P. gingivalis between group 1 and group 3 (median: 12, interquartile range: 6 to 29.5) did not reach a statis tically significant level (dif ference: 19.05; P >0.05). The number of implants that had an FAI microgap contaminated with A. actinomycetemcomitans and P. gingivalis acc ording to the different implant groups is presented in Table 4 2. Three of ten implants of group 1 had FAI microgaps colonized

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51 by A. actinomycetemcomitans whereas none of the implants of this group had FAI m icrogaps colonized by P. gingivalis In contra st, 10 of 10 implants in group 2 and nine of 10 implants in group 3 had FAI microgaps colonized by both A. actinomycetemcomitans and P. gingivalis There was a statistically significant difference for the number of implants that had FAI microgaps colonized by A. actinomycetemcomitans between groups 1 and 2 (x 2 = 10.76; P <0.05) and be tween groups 1 and 3 (x 2 = 7.5; P <0.05). Similarly, there was a statistic ally significant difference be tween groups 1 and 2 (x 2 = 20; P <0.05) and between groups 1 and 3 (x 2 = 16.36; P <0.05) regarding the number of implan ts that had FAI microgaps colo nized by P. gingivalis Table 4 1. Median number of colony forming units (interquartile range) for A. actinomycetemcomitans and P. gingivalis by implant g roup Group A. actinomy cetemcomitans (CFU) P. gingivalis (CFU) 1 (n=10) 0 (0 to 1) 0 (0 to 0) 2 (n=10) 81 (44.5 to 96.5) 55 (35.5 to 96) 3 (n=10) 24.5 (11 to 56.5) 12 (6 to 29.5) P <0.05; group 1 versus group 2 for A. actinomycetemcomitans (Kruskal Wallis test with Dunn comparisons). actinomycetemcomitans (Kruskal Wallis test with Dunn comparisons). gingivalis (Kruskal Wallis test with Dunn comparisons). T able 4 2 Number of imp lants with a fixture abutment interface microgap c ontaminated with A. actinomycetemcomitans and P. gingivalis by implant g roup Group Number of Implants Contaminated With A. actinomycetemcomitans Number of Implants Contaminated With P. gingivalis 1 (n=10) 3 0 § 2 (n=10) 10 10 3 (n=10) 9 9 P <0.05; group 1 versus group 2 for A. actinomycetemcomitans (x 2 test). actinomycetemcomitans (x 2 test). gingivalis (x 2 test). § P <0.05; group 1 versus group 3 for P. gingivalis (x 2 test).

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52 CHAPTER 5 DISCUSSION The present study shows that the test ed dental implants with a Morse taper internal connection had negligible bacterial penetration down to the thr eaded part of the FAI under in vitro conditions compared to that of a tri channel internal connection The Morse t aper is a method used by machinists to reliably join two rotating machine components. The principle of the Morse taper is that of the cone i n the cone. The trunnion (the male portion) and the bore (the female portion) are both uniformly tapered. When the trunnion of the abutment is tapped or screwed into the bore of the implant fixture they come into intimate contact. The conical taper compr esses the walls in the bore as it expands. Thus, the stresses inside the materials keep both components fixed together 151 The orthopedic industry has adapted these tapers, under the generic name of Morse tapers, as a means of reliably joining modular components of total joints directly on the operation table 152 Fi gure 5 1 Internal Morse taper connection with threaded solid abutment fixation 153

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53 The Morse taper loc k guarantees a superior mechan ical stability compared to the external hexagonal connections, or butt joint design. 154 155 T his results in a better short and long term clinical perfor mance. 153 156 158 I n a recent 4 year prospective clinical study on 1,920 Morse taper connection implants used in different prosthetic applications high survival (97.5%) and success rates (96.6% ) were reported. A mean distance from bone crest to implant shoulder of 1.07 mm and very few prosthetic complications at the implan t abutment interface (0.65%) was found 156 In an 8 year study on 275 single tooth restorations with Morse taper connecti on implants, Doring et al. (2004) r eported an implant survival rate of 98.2%, with no mechanical complications associated with the prost hetic co mponents at the implant abutment interface. 153 In another similar study on single tooth Morse taper connection implants with a mean fol l ow up period of 6.3 years, Weigl f ound a very low percentage (1.3%) of abutment loosening. 158 These re sults were confi rmed by a recent study on 307 Morse taper connecti on implants, with a four year fol low up, where high survival (98.4%) and success rates (97.07%) were reported, with a mean distance from bone crest to implant shoulder of 1.14 mm and a very l ow incidence of mechanical com plications (0.66% abutment loosening). 159 T he results of these studies are in accordance with previous work on Mo rse taper connection implants, in which the use of tapered abutment connection, providing high resistance to bending and rotational forces during clinical function, reduced the risk of abutment loosening at the implant abutment inter face. 157 160 Features of the implant abutment conne c tion were considered to influ ence not only the mechanical behavior, but also the biologic behavior of implants. 160 S tability of the implant abutment connection has been addressed to eliminate screw loosening, but

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54 also to distribute load more favorably in bone. 154 155 160 T he effect of implant abutment design on marginal bone level is, however, highly debatable. 160 161 S ome authors have suggested that micro movements at the implant abutment inter face could lead to bone resorption. 162 163 This hypoth esis still has to be tested, but Morse taper connection implants can certainly avoi d micro movements at the implant abutment interface, preventing crestal bone loss around im plants. 14 M arginal bone stability has always been considered one of the most important reference criteria to evaluate implant success over time. 50 Some authors have advocated that a higher bacte rial contamination may be rel ated to a misfit at the im plant abutment in terface caused by screw loosen ing. 164 165 S crew loosening can damage interfa ces in implant components, favoring contamination of their internal parts by microorganisms. Bacterial leakage between implants and abutments occurs and this leakage is higher wh en the abutment screw is tight ened and loosened repeatedly. 164 165 F or these r easons, the Morse taper implant abutment connection could provide an efficient seal against m icrobial penetra tion, significan tly reducing the microgap dimensions at the implant abutment interface, and contributing to a minimal level of peri implant tissue inflammation 166 W ith Morse taper connection implants, the gap is clos ed so tightly that the abut ment and the fix ture behave like a single piece. F or this reason, there is effectively no mi crogap and no bacterial leakage. 166 W ith the ta pered interference the abutment emergence geometry leads to 89 167 L azzara and Porter w ere the first auth ors to discover that the place ment of platform switched implants resulted in a smaller vertical change in the crestal bone level than was typically se en when restoring conventional

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55 im plants with abutments of matching diameter. 89 T he bio logic ration ale of the platform switch ing design or horizontal off set at the implant abutment interface is actually explained as the consequence of the hori zontal repositioning of the microgap. 167 168 B asically, the principle involv ed is to distance the abutment fixture microgap aw ay from the bone as far as pos sible. This is very important, because the microgap h ar bors bacteria that produce toxins; if bacteria are more distant from th e bone, it is subsequently pos sible to minimize bone loss. 89 166 170 Three of our 10 implants with this Morse taper connection (group 1) had one CFU of A. actinomycetemcomitans In addition, none of those implants developed CFUs for P. gingivalis These results seem to be relevant wi th the g eometry of the internal connection ; because nine of 10 implants with a tri channel inter nal connection (group 3) devel oped multiple CFUs for both A. actinomycetemcomitans and P. gingivalis However, there was no statistically significant difference between implants of groups 1 and 3 regarding the number of CFUs of P gingivalis Microbial penetration along the internal part of dental i mplants was reported in some in vitro studies using implants with different geometries of the FAI. 8 16 18 For ins tance, Quirynen et al. (1994) demon strated that bacterial in vasion of the FAI microgap was detected when fixtures and abutments were as sembled and installed in a liquid blood medium inoc ulated with oral microorganisms. 8 Similarly, Jansen et al. (1997) reported microbial leakage of 13 differ ent implant abutment combinations using E. coli as the indicator bacteria. 18 In addition, an in vivo study by Quirynen and van Steenberghe (1993) reported the pres ence of microorganism s in the inner threads of ext er nal hex implants. 7 All screw threads in this study harbored significant quantities of

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56 microorganisms. Most recently, Callan et al. (2005) described moderate to hi gh levels of eight different p eriodontopathogenic microorgan isms, including A. actinomycetemcomitans and P. gingivalis colonizing the FAI using DNA probe analysis. 9 Interestingly, the study did not detec t the colonization of the screw threads of the abutments. This is in contrast to what was found in the present study, where the threads of the abutme nts of groups 2 and 3 were col onized with bacteria. This difference may lie in the sample collection technique. Callan et al. (2005) used paper points for sample collection, whereas in th e present study, sterile calcium alginate swabs were used for the microbial sampling. The calcium alginate swabs have a more brush like appearance compared to the paper points, which may allow a more intimate contact with the threads of the abutment compared to the paper points. In addition, our group used CFUs, wh ereas Callan et al. (2005) used DNA probe analysis This method of direct plating, being less technique sensitive than the DNA probe analysis, might help explain the differences in regards to the abutment thread sampling. A recent study showed increases in probing pocket depth, clinical inflammation and numbers of periopathogens seem to indicate that a local bacterial driven inflammatory reaction may be responsible for the tissue destruction seen at failing implants. 171 In the present study, we tested for microbial coloni zation of the FAI mi crogap by A. actinomycetemcomitans and P. gingivalis because both microorganisms have an established rol e as putative period ontal path ogens. 172 In this context, the bacte rial flora associated with peri implantitis resembles that of chronic peri odontitis w ith significa nt levels of bacteria such as Fusobacterium spp., Treponema spp., Tannerella forsythia (previously T. f orsythensis), Prevotella intermedia, A. actinomycetemcomitans

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57 and P. gingivalis 116 118 An FAI that is coloni zed early by putative periodon tal pathogens such as A. actinomycetemcomitans and P. gingivalis may act as a reservoir of bacteria. This contributes t o the establishment and mainte nance of microflora that resembles that of chronic periodontitis. In fact Quirynen et al. (2006) using a check erboard DNA DNA hy bridization and real time poly merase chain reaction, revealed that a complex microbiota with se ve ral pathogenic species was es tablished in peri implant pockets within 2 weeks after abutment connection. 5 However, the mere presence of putative periodontal pa th ogens does not indicate a di rect etiologic relations hip that may lead to a destruc tive process but may simply indicate a potential pathogenic environment. 173 It is generally accepted that higher levels of bacteria must be present for extended periods of time to cause tissue damage 174 175 However, coloniz ation of periodontal pathogens above threshold levels significantly increased the probability for subjects to have deep pockets or progressive disease. 176 In patients with peri implantit is, bacterial cell samples were found at stable and diseased implant sites, indicating that a total increase in the bacterial burden was present in patients with peri implantitis 171 A specific microbiological profile has been found comparing healthy and diseased implant sulci. Failing implant sites have demonstrated an infection characterized by microbial species similar to those in periodontitis 107 113 114 177 In these patients no distinct differences were seen between healthy and disease d sites. Periodontitis associated microflora was foun d at stable and f ailing implant sites in a study by Leonhardt et al. (1999); Staphylococcus spp enterics and Candida spp ., were found in 55% of the 37 patients with peri implantitis lesions. 116 This study also showed a significantly higher number of samples positive for A. actinomycetemcomitans in

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58 patients with peri implantitis than in healthy controls. The patients with peri implantitis were found to have a microbiological profile of adult periodontitis at both healthy and diseased sites. The total bacterial burdens together with other factors such as loading, anatomical and local host response may contrib ute to the destructive process in peri implantitis. Few studies focused on the decontamination of the inner implant cavity of two stage implants. 178 180 In a recent study, Paolantonio et al. (2008) reported that the application of a 1% chlorhexidi ne gel in the inter nal part of the fixture before abutment placement and screw tightening coul d be an effective method to re duce bacterial colonization over a 6 mont h period. 179 The authors reporte d their findings for dental im plants with an external hex design that was previ ously shown to exhibit microbial leakage at th e FAI microgap. 7 8 In addition, Groenendijk et al. (2004) re ported that, the internal implant decontamination with 0.2% chlorhexidine solution led to a reduced gin gival index and crevicul ar fluid flow compared to sa line treated controls. 178 Although, the clinical impact of bacterial leakage on the implant survival rate seems to be very limited, as shown by longitudinal and cross sectional studies, 181 the exclusion of bacteria from peri implant regenerative procedures is con sidered of paramount importance to obtain clinical success and avoidance of peri implant disease 182 Peri implantitis may ha ve a multifactorial background however, the increase and maintenance of bacteria in the FAI may prove to show a hyper inflammatory trait in patients set ting off the initiation of tiss ue destruction around implants in a few patients. Loading forces on implants may also contribute to the bacterial colonization of the FAI microgap. One disadvantage of the present in vitro study is that loading

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59 condi tions were not applied. For in stance, i n an in vitro experiment using load ing forces, Steinebrunner et al. (2005) evaluated bacterial leakage along the FAI microgap and discovered statistically significant differences between five implant systems with respect to the nu mber of chewing cycles and bac terial colonization. 16 Thus, it is important to confirm or contrast the results of the present study using loading conditions. The importance of the position, size, and geome try of the implant on marginal b one levels was a sub ject of various studies demonstrating that several factors are important regarding peri implant marginal bone loss. 13 14 130 131 The bacterial colonization of the FAI microgap was r eported to be one of these fac tor s. The potential col onization of oral microorgan isms of the FAI microgap is presumably impacted by multifactor condit ions like the precision fit be tween the implant components, torque forces when the components are connected, and loading forces when the im plants are in function. Indeed, Zipprich et al evaluated the dynamic behavior of dental im plants with different designs of the fixture abutment connection with respect to microbial colonization. The authors reported the micromovement of the fixture abutme nt complex of implants loaded at an angle of 30 when a force of up to 200 N was ap plied. Interestingly, the same implant system used in our experiment was one of four systems reported to exhibit no micromovement when loaded at 100 N and one of two systems showing no measur able microgap when loaded at 200 N. 138 The authors speculated that cert ain implant designs would mini mize the pumping effect between the fixture and the abutment, thus preven ting bacterial colonization of the FAI interface. The present study indicated that differences in im plant design may affect t he potential risk for coloniza tion of oral microorganisms into the FAI microgap. Also, this

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60 study indicat ed a negligible bacterial pene tration down to the threaded part of the FAI of dental implants with a Morse taper connection

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61 LIST OF REFERENCES 1. Cochran DL, Hermann JS, Schenk RK, Higginbottom FL, Buser D. Biologic width around titanium implants. A histome tric analysis of the implanto gingival junction around unloaded and loaded nonsubmerged implants in the canine mandible. J Periodontol 1997;68(2):186 98. 2. Tarnow DP, Cho SC, Wallace SS. The effect of inter implant distance on the height of inter implant bone crest. J Periodontol 2000;71(4):546 9. 3. Mombelli A, Buser D, Lang NP. Colonization of osseointegrated titanium implants in edentulous patients. Early results. Oral Microbiol Immunol 1988;3(3):113 20. 4. van Winkelhoff AJ, Goene RJ, Benschop C, Folme r T. Early colonization of dental implants by putative periodontal pathogens in partially edentulous patients. Clin Oral Implants Res 2000;11(6):511 20. 5. Quirynen M, Vogels R, Peeters W, van Steenberghe D, Naert I, Haffajee A. Dynamics of initial subging ival colonization of 'pristine' peri implant pockets. Clin Oral Implants Res 2006;17(1):25 37. 6. Furst MM, Salvi GE, Lang NP, Persson GR. Bacterial colonization immediately after installation on oral titanium implants. Clin Oral Implants Res 2007;18(4):50 1 8. 7. Quirynen M, van Steenberghe D. Bacterial colonization of the internal part of two stage implants. An in vivo study. Clin Oral Implants Res 1993;4(3):158 61. 8. Quirynen M, Bollen CM, Eyssen H, van Steenberghe D. Microbial penetration along the impl ant components of the Branemark system. An in vitro study. Clin Oral Implants Res 1994;5(4):239 44. 9. Callan DP, Cobb CM, Williams KB. DNA probe identification of bacteria colonizing internal surfaces of the implant abutment interface: a preliminary study J Periodontol 2005;76(1):115 20. 10. Ericsson I, Persson LG, Berglundh T, Marinello CP, Lindhe J, Klinge B. Different types of inflammatory reactions in peri implant soft tissues. J Clin Periodontol 1995;22(3):255 61. 11. Persson LG, Lekholm U, Leonhardt A, Dahlen G, Lindhe J. Bacterial colonization on internal surfaces of Branemark system implant components. Clin Oral Implants Res 1996;7(2):90 5. 12. Hermann JS, Cochran DL, Nummikoski PV, Buser D. Crestal bone changes around titanium implants. A radiogra phic evaluation of unloaded nonsubmerged and submerged implants in the canine mandible. J Periodontol 1997;68(11):1117 30.

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62 13. Hermann JS, Schoolfield JD, Schenk RK, Buser D, Cochran DL. Influence of the size of the microgap on crestal bone changes around titanium implants. A histometric evaluation of unloaded non submerged implants in the canine mandible. J Periodontol 2001;72(10):1372 83. 14. King GN, Hermann JS, Schoolfield JD, Buser D, Cochran DL. Influence of the size of the microgap on crestal bone le vels in non submerged dental implants: a radiographic study in the canine mandible. J Periodontol 2002;73(10):1111 7. 15. Piattelli A, Vrespa G, Petrone G, Iezzi G, Annibali S, Scarano A. Role of the microgap between implant and abutment: a retrospective h istologic evaluation in monkeys. J Periodontol 2003;74(3):346 52. 16. Steinebrunner L, Wolfart S, Bossmann K, Kern M. In vitro evaluation of bacterial leakage along the implant abutment interface of different implant systems. Int J Oral Maxillofac Implants 2005;20(6):875 81. 17. Buser D, Mericske Stern R, Bernard JP, Behneke A, Behneke N, Hirt HP, et al. Long term evaluation of non submerged ITI implants. Part 1: 8 year life table analysis of a prospective multi center study with 2359 implants. Clin Oral Im plants Res 1997;8(3):161 72. 18. Jansen VK, Conrads G, Richter EJ. Microbial leakage and marginal fit of the implant abutment interface. Int J Oral Maxillofac Implants 1997;12(4):527 40. 19. Leventhal GS. Titanium, a metal for surgery. J Bone Joint Surg Am 1951;33 A(2):473 4. 20. Beder OE, Stevenson JK, Jones TW. A further investigation of the surgical application of titanium metal in dogs. Surgery 1957;41(6):1012 5. 21. Albrektsson T. The response of bone to titanium implants. Critical Reviews in Biocompat ibility 1984(1):53 84. 22. Davis DM. The shift in the therapeutic paradigm: osseointegration. J Prosthet Dent 1998;79(1):37 42. 23. Sconzo J. The complete mandibular subperiosteal implant: an overview of its evolution. J Oral Implantol 1998;24(1):14 5. 24. Linkow LI, Giauque F, Ghalili R, Ghalili M. Levels of osseointegration of blade /plate form implants. J Oral Implantol 1995;21(1):23 34. 25. Zarb GA. A status report on dental implants. J Can Dent Assoc 1983;49(12):841 3.

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63 26. Branemark PI, Hansson BO, Ade ll R, Breine U, Lindstrom J, Hallen O, et al. Osseointegrated implants in the treatment of the edentulous jaw. Experience from a 10 year period. Scand J Plast Reconstr Surg Suppl 1977;16:1 132. 27. Adell R, Eriksson B, Lekholm U, Branemark PI, Jemt T. Long term follow up study of osseointegrated implants in the treatment of totally edentulous jaws. Int J Oral Maxillofac Implants 1990;5(4):347 59. 28. Albrektsson T, Dahl E, Enbom L, Engevall S, Engquist B, Eriksson AR, et al. Osseointegrated oral implants. A Swedish multicenter study of 8139 consecutively inserted Nobelpharma implants. J Periodontol 1988;59(5):287 96. 29. Jemt T. Modified single and short span restorations supported by osseointegrated fixtures in the partially edentulous jaw. J Prosthet Dent 1986;55(2):243 7. 30. Branemark PI, Adell R, Breine U, Hansson BO, Lindstrom J, Ohlsson A. Intra osseous anchorage of dental prostheses. I. Experimental studies. Scand J Plast Reconstr Surg 1969;3(2):81 100. 31. Albrektsson T, Branemark PI, Hansson HA, Lin dstrom J. Osseointegrated titanium implants. Requirements for ensuring a long lasting, direct bone to implant anchorage in man. Acta Orthop Scand 1981;52(2):155 70. 32. Esposito M, Hirsch JM, Lekholm U, Thomsen P. Biological factors contributing to failure s of osseointegrated oral implants. (I). Success criteria and epidemiology. Eur J Oral Sci 1998;106(1):527 51. 33. Zarb GA, Albrektsson T. Consensus report: towards optimized treatment outcomes for dental implants. J Prosthet Dent 1998;80(6):641. 34. Laney WR. In recognition of an implant pioneer: Professor Dr. Andre Schroeder. Int J Oral Maxillofac Implants 1993;8(2):135 6. 35. Berglundh T, Abrahamsson I, Lang NP, Lindhe J. De novo alveolar bone formation adjacent to endosseous implants. Clin Oral Implants Res 2003;14(3):251 62. 36. Cardaropoli G, Araujo M, Lindhe J. Dynamics of bone tissue formation in tooth extraction sites. An experimental study in dogs. Journal of clinical periodontology 2003;30(9):809 18. 37. Lang NP, Hammerle CH, Bragger U, Lehmann B, Nyman SR. Guided tissue regeneration in jawbone defects prior to implant placement. Clinical oral implants research 1994;5(2):92 7.

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76 BIOGRAPHICAL SKETCH Dr. Michael Tesmer grew up in Florida, moving from Tampa to Port Richey at the start of high school. After which, he attended Florida Gulf Coast University where he graduated in the spring of 2004. He received his Doctor of Dental Medicine degree in the summer of 2008 from the University of Florida. Michael completed his post doctoral residency in periodontology at the University of Florida in spring 2011. After graduation, Dr. Tesmer continues to contribute to the field of p eriodontics through clinical practice, investigational research, academic instruction, and lecturing