Unravelling the effect of macro and microscopic design of dental implants on osseointegration: a randomised clinical study in minipigs

1 UNRAVELING THE EFFECT OF MACRO AND MICROSCOPIC DEGISN OF DENTAL IMPLANTS ON OSSEOINTEGRATION: A RANDOMISED CLINICAL STUDY IN MINIPIGS Authors: Ríos-Santos JV 1, Menjívar-Galán AM2 Herrero-Climent M3, Ríos-Carrasco B4, Fernández-Palacín A5, Perez RA6, Gil FJ7. 1 Ríos-Santos JV. Tenured lecturer of Advanced Periodontics. University of Seville, Spain. 2 Menjívar-Galán AM. Department of Periodontology, University of Seville, Spain. 3 Herrero-Climent M. Private Practice, Porto Dental Institute, Portugal. 4 Ríos-Carrasco B. Department of Periodontology, University of Seville, Spain. 5 Fernández-Palacín A. Department of Statistics, University of Seville, Spain. 6 Perez RA. Bioengineering Institute of Technology, Universitat Internacional de Catalunya, Spain. 7 Gil FJ. Bioengineering Institute of Technology, Universitat Internacional de Catalunya, Spain. *Corresponding author: Gil FJ School of Dentistry. Universitat International de Catalunya, Barcelona , Spain C/ Inmaculada 22 08017 ,Barcelona. Spain +34 932541800 [email protected] 2 ABSTRACT: Several dental implants are commercially available and new prototype design are constantly being fabricated. Nevertheless, it is still unclear what parameters of the design affect most the osseointegration of dental implants. The purpose of this study is assess the effects of the microscopic and macroscopic design of dental implants in the osseointegration by comparing 3 macroscopic designs (Straumann Tissue Level (STD), Essential Cone (ECD) and Prototype Design (PD)) and 6 surface treatments (SLA, SLActive, ContacTi, Shotblasting and 2 test surfaces). A total of 96 implants were placed in 12 minipigs. The implant stability quotient (ISQ), was assessed at the time of implantation, as well as at 2, 4 and 8 weeks. Histomorphometric and statistical analyses were conducted at the different sacrifice times, being 2, 4 and 8 weeks, to analyse the bone to implant contact (BIC), the Bone Area density (BAT) and the density of bone outside the thread region (ROI). The macroscopic design results showed higher ISQ values for the ECD, whereas the histomorphometric analysis showed higher ossoeintegration values for the STD. Regarding the microscopic design, both SLActive and ContacTi showed superior results in terms of osseointegration and reduced the osseointegration times from 8 weeks to 4 weeks compared to the other analyzd surfaces. In conclusion, each of the macroscopic and microscopic designs need to be taken into account when designing novel dental implants to enhance the osseointegration process. Keywords: dental implants, implant surfaces, macrodesing and microdesing, osseointegration, implant desing. 3 1.INTRODUCTION One of the most influential factors in implant osseointegration is the morphology and surface of the implant. Nowadays, research is striving to design dental implants that achieve the highest percentage of bone to implant contact (BIC) and an optimized distribution of stresses under applied loads 1 . On the one hand the macrodesign of the dental implant is designed to transfer the applied load in an homogenous manner and hence reduce bone resorption, as well as is in charge of offering a high surface are in contact with the host bone. On the other hand, the microdesign of the implant, which refers to the surface morphology of the implant, is in charge of offering an increased specific surface area by presenting cavities and pits that allow bone to grow within these pores of tens of microns and hence enhance the osseointegration 2 . Regarding the macroscopic design, there are several features that define the threads and it has become of great importance controlling each of the features to design an optimized implant. For instance, the apical face angle, which is the angle formed between the most apical angle of the thread and the perpendicular plane to the axial axis of the implant itself has been vastly studied in previous works and has been shown to have greatest influence in dispersing the forces to the highest extent. Furthermore, as deduced from the structure, as the apical face angle becomes smaller, the placement of the dental implant is highly favoured. Along with the apical face angle, previous studies regarding the thread pitch, which refers to the distance between adjacent threads, previously showed that the dental implant presenting higher thread pitch increased the stability. The enhanced stability in the presence of bigger thread pitches allows decreasing the implant size. Other parameters, such as the shape of the thread or the apex width also play a pivotal role in ensuring an optimal integration with the host bone 3 . Besides the overall morphology of the implant, the surface of the implant plays a key role at the cellular level. Cellular interactions are mediated by the initial cell adhesion to the surface, which are regulated by the topographical and physicochemical features. In general, increased surface roughness have been shown to lead to higher protein adsorption and hence allow increased cellular interactions 4 . Surface properties of the implant hence play a decisive role in the short term and have a significant role in the final osseointegration, therefore is considered as an important feature to take into account when designing dental implants 5 . Sand blasted implants are achieved by blasting surfaces by small particles (TiO2, SiO2,Al2O3, etc) The resulting surface topography is usually anisotropic consisting of craters and ridges. By developing moderately rough surfaces through sandblasting and acid etching, result in uniform roughness with pits and 4 craters of micrometric size, to an increase the surface area. Previous works have shown a faster osseointegration of dental implants, characterised by the formation of new bone, which results in more predictable immediate and early loading procedures 6 - 7 . The first bioactive surface to be created was called SLActive (Institut Straumann AG, Basel, Switzerland). It is a modified version of the classic surface obtained by sandblasting and acid etching (SLA - Institut Straumann AG, Basel, Switzerland), which was introduced in a nitrogen atmosphere to avoid contamination of the passive elements in the air. It was then stored in an isotonic saline solution. By doing this, scientists were able to change the surface tension, obtaining more negatively surface energy and a higher wettability4.This surface has shown excellent clinical results in both clinical and experimental studies compared to its predecessor 8 - 9 . Coating the implant surface with a layer of CaP (Carbonite-containing apatite) to achieve bioactivity has attracted much interest from researchers, who have demonstrated that it accelerates early bone healing and bone bonding for its rapid protein absorption and the osteoconductivity of the implant. The technique described by Kokubo in 1996 led to the surface development called ContacTi (Klockner Implant System. SOADCO, Andorra). The technique consisted of an alkaline immersion process, a thermal treatment followed by a biomimetic treatment consisting of an immersion in simulated body fluid. By doing this, the titanium surface was coated with a layer of apatite that interacted with the underlying bone accelerating the osseointegration process. The ContacTi surface was obtained from a two-step surface treatment. The surface was shot-blasted or bombarded with alumina particles (Al2O3), which provided an optimum microroughness for adhesion, proliferation and differentiation of human osteoblastic cells, and then given a subsequent treatment (alkaline immersion and thermal treatment). The metal surface changes with the thermochemical process, which transforms it from bioinert to bioactive, thanks to the formation of a dense and amorphous sodium titanate gel. The surface acquires bioactivity and demonstrates in vitro the formation of a layer of hydroxyapatite by crystallisation when introduced in a simulated body fluid 10 . Given the importance of the morphology of implants and their surfaces -and since it is accepted that rough surfaces are the most suitable to shorten treatment times, we believe there is justification to test these different surfaces given that they can have a positive effect on healing. The purpose of the study is to assess the influence of microscopic and macroscopic implant design on osseointegration. 2.MATERIAL AND METHODS 5 Implant description The study was designed to assess the influence of different implant surfaces and morphologies on bone healing. Eight implants with different morphologies and surfaces were used as described in Table 1. Macro Design In order to assess the effect of the overall morphology on the bone healing ability, the studied morphologies are the Straumann Tissue Level, Klockner Essential Cone and implant prototype. The macrodesign of the Straumann Tissue Level implant -with its parallel-walls- features an 18º V-shaped thread with a 1.25 mm thread pitch and a 0.1 mm apex width. The morphology of the Essential Cone Implant is characterised for being a conical implant with a 45º V-shaped thread, a 2.2 mm thread pitch with two inputs to increase the insertion speed and apex width of 0.21 mm. The morphology of the prototype implant is similar to the Klockner Essential Cone implant but differs in a 2-tenth reduction in the implant core, a sharper thread and the apex width is 0.12 mm (Figure 1). The different implant morphologies were compared presenting the same surface treatment, which was the Shotblasting. Micro Design The different studied surfaces were the SLA, (Sandblasted surface plus acid eatched) (Sa/roughness 0.96±0.07), SLActive (Sandblasted surface plus acid etching in a nitrogen atmosphere) (Sa/roughness 1.69±0.03) 11 , Shotblasting (Sandblasted surface with alumina oxide plus acid passivation) (Sa/roughness 3.1±0.4), ContacTi (shot-blasted or bombarded with alumina particles (Al2O3) and posterior alkaline immersion and thermal treatment) (Sa/roughness 3.5±0.3) 12 , Test surface 1 (sandblasted surface plus acid attack) (Sa/roughness 2.30±0.18), Test surface 2 (sandblasted surface plus acid attack in a argon environment) (Sa/roughness 2.67±0.39). Roughness was evaluated for the test surfaces in the framework of the recommendations by Wennerberg and Albrektsson (2000) on topographic evaluation for dental implants. A white light interferometer microscopy (Wyko NT1100, Veeco) was used. The surface analysis area was 459.9 × 604.4 µm2 for all the microrough surfaces. Data analysis was performed with Wyko Vision 232TM software (Veeco, USA). A Gaussian filter was used to separate waviness and form from the roughness of the surface. Cut-off values, λc = 0.8 mm, for micro-rough surfaces were applied. The measurements were made in three different surfaces of each type of surface treatment to characterize the Ra (the average roughness), which is the arithmetic average of the absolute values of the distance of all points of the profile to the mean line. Animal surgery 6 The study was performed in a reduced number of animals 8- 13 .Twelve 6-year-old female minipigs provided by the University of Córdoba, Centralised Experimental Animals Service, Rabanales Campus with cattle farms registration number ES14021000000933 authorised in favour of Mr Nahúm Ayala and Mr Ríos-Santos, project number 20-08-15-293 with the approval of the Ethics Committee of the Junta de Andalucía were used. All the requirements for animal testing were met, in compliance with the regulations in force in Spain and the European Union. Animals were fed a diet of powdered and liquid food throughout the study, undergoing oral prophylaxis using an aseptic technique three weeks before the experimental surgeries. The feeding of the animals was suspended the night before the surgery. The animals were pre-anaesthetised with Xylazine Hydrochloride and Ketamine, and maintained on gas anaesthesia (Isoflurane at 5% - Oxygen). They were kept hydrated during surgery by administering Ringer Lactate’s solution intravenously. The animal’s heart and breathing rates were also monitored. Instructions recommended by the manufacturers were followed at all time for implant insertion. The study consisted of two surgical procedures, performed on maxillas of the minipigs. The 12 minipigs were randomly divided into three groups, placing eight implants in the maxillas of each pig, one of each type. First surgical procedure During the first surgery, the posterior teeth of the maxilla, bicuspids and molars were carefully extracted under general anaesthesia, by making an incision in the premolar and molar regions in both maxillary sides. At least six-months healing was allowed in order to achieve a fully mature alveolar crest in this area of the maxilla and to ensure complete healing of the places from where the teeth had been extracted. The area was closed by primary intention with interrupted dermal sutures using synthetic polyamide (Supramid® 4-0). After the surgery was completed, Buprenorphine hydrochloride was administered for pain control and amoxicillin to prevent infection. Animals were continuously monitored to prevent oedema formation, wound dehiscence and infection. Second surgical procedure After these 6 months of healing, 8 implants (one of each group) were randomly placed in the maxilla of each pig, which had previously had all their teeth removed in the first surgical procedure. The implants were placed following the manufacturer’s recommendations, namely, the submerged technique, being 7 completely covered by the skin. At the end of the preparations, the depth was checked to ensure that the polished neck was placed supracrestal and that the separation between them was at least 4 mm. Closing taps were placed on the implants and after irrigating them with sterile saline solution, the area was closed by primary intention with interrupted dermal sutures using synthetic polyamide (Supramid® 4-0). After the surgery was completed, Buprenorphine hydrochloride and amoxicillin were administered, followed by a continuous monitoring as previously described. Animal sacrifice At the specified time points, animal sacrifice was performed by an overdose of sodium pentothal in perfusion through the carotid arteries. This solution also contained a mixture composed of glutaraldehyde at 5% and formaldehyde at 4% at a pH of 7.2. Each group was sacrificed at a different time point from the second surgery, being 2, 4 and 8 weeks, respectively. Analysis of in vivo results Once the implants were placed, the implant stability quotient (ISQ) was measured by means of Resonance Frequency Analysis (RFA) using the Osstell ISQ® (Gothenburg, Sweden) according to the manufacturer's indications. Similarly, RFA measurements of the implants were recorded at the time of sacrifice. In order to carry out the analysis, block sections -including the implant, alveolar bone and mucous membranewere obtained from each animal. The maxillae under study were cut using an Exakt 310® (Exact Vertriebs, Norderstedt, Germany) oscillating saw to delimit the implant and the surrounding bone with a thickness of no more than 4 mm to ensure a correct posterior fixation. Samples were then immersed in 10% buffered formalin solution for at least two weeks prior to processing. Once the samples were fixed with formalin, samples were dehydrated in graded ethanol solutions (70%, 80%, 96% and 100%). The dehydrated samples were infiltrated and embedded in methyl methacrylate (MMA) resin (Technovit 7200 VLC, Kulzer-Heraus, Germany). Each polymerised block was cut in the axial direction of the implant with a diamond band saw to avoid overheating and deteriorating the tissues surrounding the implant. All sections were polished with the automatic polishing machine (Exakt 400 CS, Exakt, Germany). 8 The polished cuts were examined under scanning electron microscope (SEM, JSM-6400, Jeol, Japan) using scattered retro electrons to further differentiate the bone from the surface. Images at different magnifications were obtained. The histological study was carried out in such a way that the histopathologist in charge of the histomorphometric study was unaware of the sample type in each animal being assessed. Three main parameters were then analysed:  BIC: The percentage of direct contact between the mineralised bone and the titanium surface (Bone-implant contact).  BAT: The area of mineralised bone relative to the total tissue in the area between two threads.  ROI: Density of the bone in the outer area delimited by the beginning of the implant threads and one millimetre depth. Statistical analysis The statistical analysis was carried out by the Statistics Department of the Faculty of Dentistry, University of Seville, using the IBM SPSS 24.0 statistical package for Windows. Mean values and standard deviations were calculated globally and by groups (surface area, surface area of each moment, etc…) A two-factor ANOVA was applied to compare numerical variables between the groups. When these tests were significant, multiple comparisons tests (Bonferroni) were used to detect the groups that differed and to obtain confidence intervals at 95% to quantify these differences. The significance of the differences was established for p <0.05. 3.RESULTS A total of 96 implants were placed in the maxillae of twelve minipigs. The BIC, BAT, ROI and the ISQ evolution were analysed after 2, 4 and 8 weeks. The histological and histomorphometric analyses revealed that the samples showed a normal macroscopic anatomy for all time points and conditions studied. On the day of the necropsy, normal bone tissue was observed in contact with all implants, with no swelling, oedema, fibrosis or mobility around them or any other complication. No changes in tissue morphology were observed, as well as no signs of increased inflammatory cell count or necrosis. Clinically speaking, the implants appeared to have osseointegrated, which was then followed by the sample processing steps. 3.1. Macro Design 9 Initially, we assessed the macroscopic implant design on the osseointegration outcome (table 2). According to the ISQ values at 2, 4 and 8 weeks, (table 3) the different implants presented similar values at the time of surgery, not presenting significant differences among them. This trend continued in a similar way after 2 and 4 weeks time point, although at 8 weeks the ECD presented significantly higher values than the STD. Nevertheless, there were no significant differences between the PD and the ECD and the STD. Regarding the histomorphometric analysis at the same time points, the results were slightly different. The BIC values were relatively low and not significantly different between them at 2 and 4 weeks time points, but became significantly different at 8 weeks, where the STD and PD increased the values while the ECD presented significantly lower values compared with the STD and PD. Similar trend was observed for the BAT where only the differences were significant at 8 weeks, presenting the ECD morphology significantly lower values than the STD and PD. Finally, the ROI values followed as well as similar trend, presenting similar values at 2 and 4 weeks between them, presenting significantly lower values for the ECD. Nevertheless, in this case, only ECD presented significantly lower values compared to the PD. Overall the results show that the implant morphology may present a significant effect on osseotintegration. 3.2. Micro Design We then assessed the effect of the surface morphology on the different variables (table 4). Initially we assessed the differences between the Shotblasting surface and the well-known surfaces used by Straumann, the SLA and the SLActive. In terms of ISQ values (table 5), both the SLA and the SLActive performed in an ulterior manner, presenting higher values at all-time points. Both presented significantly higher values compared to the Shtoblasting surface at 4 and 8 weeks. At 2 weeks time point, only the SLActive presented significantly higher values than the Shotblasting surface. Regarding the histomorphometric analysis, the BIC values were shown to be similar at 2 weeks, although these values became significantly higher at 4 weeks both for the SLA and SLActive compared to the Shotblasting surface. At 8 weeks, SLA and SLAactive did not significantly increase their values, whereas the Shotblasting surface significantly increased the values, not presenting significant differences among the three of them. The BAT values showed predominant values at all-time points for the SLActive, although the values were not significantly higher at any time point. Finally, the ROI values presented similar trend in all cases not showing significant differences among them. The results therefore prove the higher performance of the SLA and SLActive surface compared to the Shtoblasting surfaces. 16 8. FIGURES AND TABLES Figure 1. Macrodesign of the Straumann Tissue Level implant (1), Klockner Essential Cone implant (2) and Prototype implant (3). 17 Table 1. Morphology and surface treatment of the different implants analyzed. CODE Company Size Surface modification process Macrodesing Comments A Straumann 4.1x8mm Sandblasted + acid eatched surface Straumann Tissue Level SLA® B Straumann 4.1x 8mm Sandblasted surface + acid etching (hydrochloric/sulphuric acid) in a nitrogen atmosphere Straumann Tissue Level SLActive® C Klockner 4x8mm Shot-blasted surface with alumina particles and acid attack with hydrochloric acid Essential cone Shotblasting® D Klockner 4x8mm Shot-blasted or bombarded with alumina particles (Al2O3) and posterior alkaline immersion and thermal treatment Essential cone ContacTi® E Klockner 4x8mm Sandblasted + acid eatched surface Essential cone Test1 F Klockner 4x8mm Shot-blasted surface with alumina particles and acid attack with hydrochloric acid Straumann Tissue Level Shotblasting® G Klockner 4x8mm Sandblasted surface with alumina oxide + acid attack (hydrochloric/sulphuric acid) in argon environment Essential cone Test 2 H Klockner 4x8mm Sandblasted surface with alumina oxide + acid passivation Prototype desing Shotblasting® 18 Table 2. BIC, BAT, ROI, when comparing different implant morphologies at different time intervals. (STD: Straumann Tissue Level Design, ECD: Essential Cone Design, PD: Prototype Design) BIC (%) BAT (%) ROI (%) Mean ± Standard deviation Mean ± Standard deviation Mean ± Standard deviation 2 weeks STD 50,5±13,01 39,75±35,70 53±41,01 ECD 49,73±15,60 35,6±22,05 60,48±13,77 PD 44,4±19,02 40,85±28,28 74,42±26,62 4weeks STD 53,65±18,15 46,93±24,20 65,9±12,42 ECD 51,48±19,07 44,23±15,94 66,68±16,48 PD 36,73±23,12 34,93±21,50 66,17±14,09 8 weeks STD 70,7±14,20 67,15±11,91 70,88±27,59 ECD 47,3±3,14 50,31±2,57 56,68±5 PD 71,76±3,41 71,73±6,30 77,15±7,84 Table 3. ISQ at Surgery and ISQ at sacrifice when comparing different implant morphologies at different time intervals. (STD: Straumann Tissue Level Design, ECD: Essential Cone Design, PD: Prototype Design) ISQ Initial 2 weeks 4 weeks 8 weeks STD 70,5±7,36 68,25±8,5 69,25±3,86 68,66±4,04 ECD 70,27±6,56 72,75±5,85 70,5±5,19 76,5±3,53 PD 69,77±11,62 66,75±2,75 70,75±8,84 72,66±4,16 19 Table 4. BIC, BAT, ROI, at sacrifice when comparing different surfaces (SLA, SLActive and Shotblasting) with the same morphology Straumann tissue level. BIC (%) BAT (%) ROI (%) Mean ± Standard deviation Mean ± Standard deviation Mean ± Standard deviation 2 weeks A 53,56 ±19,80 32,4 ±12,69 41,65 ±9,58 B 61,56 ±12,77 39,52 ±9,61 47,35 ±5,78 F 50,55 ±13,01 39,75 ±35,70 53 ±41,01 4weeks A 81,6 ±12,17 57,05 ±20,38 62,05 ±14,98 B 85,36 ±13,26 67,85 ±11,84 62,66 ±17,11 F 53,65 ±18,15 46,93 ±21,33 65,9 ±12,92 8 weeks A 80,73 ±8,48 75,66 ±10,53 78,43 ±9,75 B 87,17 ±4,45 78,03 ±6,55 74,38 ±12,51 F 70,7 ±14,20 67,15 ±11,91 70,88 ±27,59 Table 5. ISQ at Surgery and ISQ at sacrifice when comparing different surfaces (SLA, SLactive and shotblasting) with the same morphology Straumann tissue level. ISQ Initial 2 weeks 4 weeks 8 weeks A 74,5 ±8,05 76,25±16,19 81±1,41 81,67±4,04 B 76,16±8,50 79,33±4,51 78,66±4,16 80,5±3,69 F 70,5 ±7,36 68,25±8,5 69,25±3,86 68,66±4,04 20 Table 6. BIC, BAT, ROI, at sacrifice when comparing different surfaces (Shotblasting, ContacTi, Test surface 1, Test surface 2) with the same morphology Essential. BIC (%) Mean ± Standard deviation BAT (%) Mean ± Standard deviation ROI (%) Mean ± Standard deviation 2 weeks C 49,73 ±15,61 35,6±35,6 60,48± 13,71 D 36,86 ±31,03 21,05±21,05 48,34±19,85 E 18,95 ±3,18 18,118,1 53,55±19,23 G 47,21 ±21,63 40,22±24,08 62,23±24,27 4 weeks C 51,48±19,07 44,24±15,94 66,68±16,48 D 83,2±8,11 61,22±11,9 58,01±10,46 E 52,23±12,88 36,54±6,85 63,29±8,40 G 70,9375±15,26 55,25±29,38 60,99±27,53 8 weeks C 47,3±3,14 50,31±2,57 56,68±5,01 D 85,58±3,81 59,53±8,67 66,56±11,24 E 64,13±27,20 53,36±29,94 63,72±24,96 G 76,85±10,53 57,04±9,32 70,85±8,4 Table 7. ISQ at Surgery and ISQ at sacrifice when comparing different surfaces (Shotblasting, ContacTi, Test surface 1, Test surface 2) with the same morphology Essential. 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