Running Title: Implant Abutment Interface Sealing

Evaluation of the implant abutment interface sealing capability of different implant –abutment connections :an in vitro study

Running title: Implant abutment interface sealing

Key wards: implant, sealing, interface, in vitro study

RadhwanHimmadiHasan(B.D.S, M.Sc., Ph.D.)*

Hussein SaadSaeed(B.D.S)**

*Assistant professor, Department of Prosthetic Dentistry, College of Dentistry, University of Mosul.E-mail:, Phone nomber:009647701632273

**General practitioner, Ministry of Health.E-mail:, Phone nomber:009647701678258

Evaluation of the implant abutment interface sealing capability of different implant –abutment connections: an in vitro study

Running title: Implant abutment interface sealing

ABSTRACT

Background and objectives: Microgap is defined as the microscopic space thatexists between the implant body and abutment.The mechanism proposed for microgap related crestal bone loss is the role of this space as a trap for bacteria Thepurpose of this study was to detect and compare bacterial microleakage at implant abutment connection of three types abutment connectionsand compare between two torques values of abutment connectionsMaterials and Methods:In this experimental studyeighteen dental implants, six external hex titanium abutments, six internal hex titanium abutments and six morse taper titanium abutments were used in this experiment. Abutmentswere fixed to implants with a torque of 20 and 30Ncmrespectively and sterilized by autoclave and each complex put in test tube containing nutrient broth, each tube was inoculated with staphylococcus bacterium and incubated at 37 C for 14 days.Then assemblies removed from the tubes, cleaned and dried and separated , a small brush used to take specimen form inside implant chamber and cultured in petri dishes containing nutrient agar then those incubated at 37 C for 24 h , after that colonies appeared identified and enumerated by using optical microscope.Statistically ,Kruskal-Whallis and Mann-Whitney test were performed to determine the significantdifference at p ≤ 0.05.Results:Kruskal-whallis test confirmed that there was no significant difference among three abutment types, Mann-Whitney test result showed significant difference between both torques (20 and 30 Ncm) of all abutment types.Conclusions:All abutment types showed bacterial microleakage, also as torque value increase the microleakage decrease for all abutment types.

Keywords:implant, sealing, interface, in vitro study, internal hex, morse taper, external hex

الخلاصة

أهداف البحث:تهدف الدراسة الى كشف وجود و مقارنة التسرب البكتيري الدقيق في المنطقة البينية بين الزرعة و الدعامة لثلاث انواع مختلفة من ارتباط الزرعة و الدعامة و كذلك المقارنة بين قيمتين من الشد للدعامات .

المواد وطرائق العمل:المواد وطرائق العمل:ثمانية عشر زرعة سنية , ست دعامات عرافة التيتانيوم الخارجية و ست دعامات عرافات التيتانيوم الداخلية و ستة دعامات التيتانيوم تفتق مورس استخدمت في هذه التجربة و قسمت الى ستة مجموعات دراسية و كل مجموعة قسمت الى مجموعتين فرعيتين ,مجموعة فرعية من ثلاث زرعات على الشد 20 و مجموعة اخرى على الشد 30.

تم قياس التسرب من الخارج الى داخل الزرعة السنية و ذلك بغمر الزرعات المعقمة مسبقا داخل انابيب اختبار معقمة و تحتوي على وسط غذائي سائل لنمو البكتريا و بعد ذلك حقن السائل ب بكترياstaphylococcus aureus ووضعهم في الحاضنة لمدة 14 يوم في درجة حرارة 37 سيليزية و من ثم اخراجها و تطهيرها من الخارج بمادة الكحول و اخذ عينات من داخل الزرعة بعد ازالة الدعامات و زرع هذه العينات في وسط غذائي أجار و من ثم قياس التجمعات البكتيرية بواسطة الميكروسكوب الضوئي

إحصائيا تم اجراء اختبار مان ويتني و كروسكال واليس لتحديد الاختلافات المعنوية عند مستوى احتمالية (0,05)

. النتائج:اظهرت النتائج انه لا يوجد فرق معنوي بين جميع انواع الارتباط بين الزرعة و الدعامات كما أظهرت النتائج ان دعامة العرافة الداخلية هي ذات التسرب الأقل تليها دعامة تفتق مورس و من ثم دعامة العرافة الخارجية الأعلى تسربا.كما أظهرت النتائج وجود فرق معنوي بين قوتي الشد 20 و 30 لجميع انواع الدعامات. الاستنتاجات: يتضح من النتائج أعلاه أن جميع انواع الدعامات أظهرت تسربا بكتيرياً و لم يكن هناك فرق معنوي. و كذلك ف ان الدعامات المشدودة على العزم 20 أظهرت تسربا أكثر من الدعامات المشدودة على العزم 30.

INTRODUCTION

Dental implant is defined as a prosthetic device made of alloplastic material(s) implanted into the oral tissues beneath the mucosal or/and periosteal layer, and on/or within the bone to provide retention and support for a fixed or removable dental prosthesis; a substance that is placed into or/and upon the jaw bone to support a fixed or removable dental prosthesis.(1)

Over the last 30years, the osseointegration phenomenon has drastically changed dental treatment restorative modalities, making dental implantology one of the most successful rehabilitation techniques among medical and dental specialties with success rates reported above 90%.(2)

Developments in dental implantology evidently changed the treatment modalities over the last 25 years. However micro-gap formation between the surfaces of implant fixture and abutment is still one of the major problems at the connection area which may lead to mechanical and biological failures such as screw loosening and periimplantitis.(3)

Microgapisdefinedasthemicroscopicspacethatexistsbetweentheimplantbodyandabutment.Themainmechanismproposedfor microgap relatedcrestalbonelossistheroleofthisspaceasatrapforbacteriaandthus,asaputativeetiologicalfactorforinflammatoryreactionintheperi-implantsofttissues.(4)

The amount of bacterial infiltration between the implants and abutments depends on factors such as the fit accuracy between the pieces and the tightening torque and micro-movements between the connected components during mastication.(5,6)

Several design alterations have been made in an attempt to reduce the implant-abutment gap, thereby decreasing both its mechanical and biological drawbacks. However, limited success has been achieved. The inclusion of polymeric components between connection parts was able to reduce but not eliminate bacterial colonization.(7,8)

Bacterial culture is a well-known method historically used to characterize the oral cavity microbiota, and considered a classical reference method in microbiology. Traditionally, culture-dependent methodologies are used to isolate, enumerate and detect probiotic organisms, especially from mixed cultures.(9)

.

Materials and Methods

Bacterial penetration experiment to measure leakage from outside to inside implant abutment connection.

Dental implants with their respetive abutments including :

a. Tixos, MC Tixos Neck Implant Internal Hex (Morse)_Leader, Italy (6 Implants 4.5 mm in diameter along with their respective abutments).

b. IMPLUS, TTS Cylindrical implant ExternalHex_Leader, Italy (6 Implants 3.75mm in diameter along with their respective abutments).

c. IMPLUS, TTS Tapered Implant Narrow Thread Internal Hex_Leader, Italy (6 Implants 3.75 in diameter along with their respective abutments).

Samples Grouping:Eighteen Implants were divided into three experimental groups(Morse taper, external hex and internal hex) and each group divided into two subgroups:

Subgroup A1 (N=3) in whichmorse taper abutments were torqued to 20Ncm, Subgroup A2 (N=3) in which morse taper abutments were torqued to 30Ncm.

Subgroup B1(N=3) in which external hex abutments weretorqued to 20Ncm, Subgroup B2(N=3) in which external hex abutments were torqued to30Ncm.

SubgroupC1(N=3) in which internal hex abutments were torque to 20Ncm and Subgroup C2(N=3) in which internal hex abutments were torque to 30 Ncm.

Preparation of broth and culture media :First the nutrient broth media was prepared by adding powder 1.3 g to 100 ml distilled water and by shaking on electrical heater until boiling ,then poured it in sterilized test tube and sterilized it by autoclave.

Also 28 g of nutrient agar powder suspended in 100 ml of distilled water and brought to the boil to dissolve completely and dispensed as required(10)

Instruments Sterilization:Each implant body and abutment and all instrument are sterilized inside surgical bags by autolavefor 15 minutes at 121 °C (250 °F) at 100 kPa (15 psi).Each implant body and abutment was unpacked under a ventilation hood usingsterile technique. (11)

Closing torque procedure:All the implants were held in an upright position on a sterileholder. This was attached to the table, with rubber ends allowing a firm tapping or screwing action, causing no damage to the implant surface. All the assemblies were manipulated under aseptic conditions.(12)

The abutment was torqued with a screw driver conneted to adigital torqometer held in upright position by the vertical arm of a dental surveyor to 20 Ncm and 30 Ncm.(12)(Figure 1).

figure-1: Closing torque procedure

Implant placement inside broth media: Each implant complex was placed in a sterilized test tube containing nutrient broth. The level of broth reached implant abutment connection level but not exceed the top opening of the abutment.(11.13)(Figure 2).

Figure-2: Implant inside broth media

Inoculation procedure:Each tube then inoculated with Staphylococcus aureus bacterium.Staphylococcus aureus(S. aureus), a major pathogen often found in the percutaneous implant-associated infections, and its adherence to the implanted surfaces was visualized and quantified.(11)(Figure 3)

Figure-3: Inoculation the broth with Staphylococcus aurius bacteria

All procedures were performed in a sterile environment, with sterile gloves and a laminar flow chamber (Valiclean, Brazil), which was covered with sterile surgical cloth (Asséptico, Brazil). All equipment was previously steam sterilized in an autoclave (Baumer, Brazil) at 121° for 15 min.(11)

Placement of tubes inside incubator: All of the test tubes were enumerated, placed in a test tube rack in a vertical position and incubated at 37°C(14).each tube was labeled according to its specific subgroup .

Post incubation proedure: After 14 days Each assembly was removed from the broth after the incubation period, carefully dried with sterile absorbent paper to remove the excess of broth. The implant-abutment interface was disinfected with 70% alcohol and dried with absorbent papers.(15,16), then rinsed dried and placed in a sterilized holder and the abutments was removed and a small sterilized brush was used to take specimen from the internal chamber of each implant.(16)

Culture proedure :Then those specimens was cultured in petri dishes containg nutrient agar and incubated at 37°c for 24 hours.

Also another specimen from each implant internal chamber was taken using small brush and diluted in test tube containing normal saline 0.1 ml and they were plated in duplicate onto nutrient-agar plates (pouring diluted bacterial solution then followed by pouring nutrient broth))13)

Counting of bacterial colonies:After 24h of incubation, the purified colonies on petri dishes were identified by means of optic microscopy to measure number of colonies of each petri dish.

Figure-4 : Bacterial colonies

Statistical Analysis:Statistical analyses were performed by SPSSat 95% level of significance ( The level of statistical significance was set at alpha=0.05.) with following tests:

1. Mann-Whitney test

2. Kruskal-Wallis test

The data obtained throughout the course of study were analyzed using Excel; Microsoft office 2010, Windows 7 Ultimate. Descriptive statistics including mean, standard deviation, minimum and maximum were calculated for each group.

RESULTS

The following results were obtained from observation of the colonies under a polarized light microscope using 10Xmagnification power.

The descriptive statistics of groups(morse taper, external hex and internal hex abutments) including mean, standard deviation were calculated (Table 1), then a comparison among samples mean of all abutment groups using Kruskal-Wallis test(Table2). According to this test, it was clear that there was no significant difference among them.(p >0.05)

Table (1): Descriptive statistics of treatment groups and control group

Table (2): Independent Samples T-test

Internal hex abutments showed less mean of bacterial colonies (leakage) followed by morsetaper and finally with external hex abutments as shown in figure (4)

Figure-4:Bacterial colonies mean of all abutment types

In order to explain the effect of the study parameter (torque values) on the mean bacterial microleakage of all abutment types, Mann-Whitney test was used for comparison between them.Table (3) illustrate the effect of torque value on mean bacterial colonies (leakage) by making comparison between sample mean for both torques (20 and 30 Ncm) of morse taper, external hex and internal hex abutments respectively and appear there is significant difference between them.(p ≤ 0.05)

Table (3):Mann-Whitney test between both torques of all abutment types

.

DISCUSSIONS

Malodor and clinical signs of bleeding after removing the cover screws or abutments may be caused by infiltration of anaerobic bacteria through the implant -abutment interface. This phenomenon of infiltration can be assessed in two ways: Verifying the passage of bacteria to the inner part of a dental implant)(17,18),or in an inverted direction.(19,20)

The degree of penetration in a specific implant system presumably is a multifactorial condition dependent on the precision of fit between the implant and the abutment, the degree of micromovement between the components, and the torque forces used to connect them(19)

The present study evaluated that there is no significant difference of bacterialmicroleakage among the three abutment types( Morse taper, external hex and internal hex). And all of them show bacterial infltration along implant abutment interface and this infiltration happened due to presence of microgaps between implant body and all abutment types. This result agrees with Jansen et al., 1997 who determined that 13 different types of prosthetic connections presented bacterial infiltration(15).This study alsoagrees withFariaet al., 2011 who showed in his study that there is no significant difference among the same three abutment types(11)

Also this agrees with Steinebrunneret al., 2005who tested five different implant systems with 8 standard implant-abutment combinations for single molar crownsbut his study differ in that he exposed the implants to a load of 1,200,000 cycles of 120 Nwith chewing simulator and this load surely increased the gaps for all implant abutment connections.(19)

Also this study disagrees with Nascimentoet al., 2012 who showed that there is significant difference among three types of abutments( Morse taper, External hex and Internal hex connections) in case of unloaded condition like our study.

But the mean of bacterial colonies was the hieghst for external hex abutments followed by morse taper abutment and finally the lowest mean for internal hex abutments and that refers that the microgap in external hex implant abutment connection is the largest followed by morse taper and internal hex which has the smallest gap. And this also agrees with Fariaet al., 2011.(11)

Also agrees with Jaworski et al., 2012who showed that morse taper and external hex systems exhibited bacterial contamination, the Morse taper implants of the system used in his study provided a better bacterial seal than external-hexagon implants of the same system(21), This is due to difference in microgaps between the two types.

The result of Mann-Whitney test between both torques (20 and 30 Ncm) of all abutment types show that there is significant difference between both torques.In contrast to the current resultSmith and Turkilmaz, 2014 showed that bacterial leakage was found in all specimens, regardless of screw torque value with titanium abutments, changing the screw torque value from 20 to 35 Ncm did not significantly affect the amount of bacterial leakage(22).By the same time it agrees with the same study butwith zirconia abutments, changing the screw torque value from 20 to35 Ncm was statistically significant.

Also our study showed that as torque increased from 20 to 30 Ncm the leakage decreased.and this agrees with Silva-Netoet al., 2012(23)

The resultwe obtained indicatethat tightening the abutments screwfrom 20 to 30 Ncm decreases the size of the microgap, which suggests amore intimate fit betweenthe implant and the abutment.

CONCLUSIONS

Within limitation of this in vitro study, the conclusions are as follows:

The seal between the implant body and theabutment could not be maintained in all abutments type which showed bacterial microleakage and this leakage was not significant between them, internal hex showed least microleakage followed by morse taper and finally with external hex showed highest microleakage.Alsot there was significant difference between both torques (20 and 30 Ncm) in all abutment types and bacterial microleakage decreased as torque increased.

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