Colonization of Helicobacter pylori in the oral cavity - an endless controversy?

Helicobacter pylori is associated with chronic gastritis, gastric or duodenal ulcers, and gastric cancer. Since the oral cavity is the entry port and the first component of the gastrointestinal system, the oral cavity has been discussed as a potential reservoir of H. pylori. Accordingly, a potential oral-oral transmission route of H. pylori raises the question concerning whether close contact such as kissing or sharing a meal can cause the transmission of H. pylori. Therefore, this topic has been investigated in many studies, applying different techniques for detection of H. pylori from oral samples, i.e. molecular techniques, immunological or biochemical methods and traditional culture techniques. While molecular, immunological or biochemical methods usually yield high detection rates, there is no definitive evidence that H. pylori has ever been isolated from the oral cavity. The specificity of those methods may be limited due to potential cross-reactivity, especially with H. pylori-like microorganisms such as Campylobacter spp. Furthermore, the influence of gastroesophageal reflux has not been investigated so far. This review aims to summarize and critically discuss previous studies investigating the potential colonization of H. pylori in the oral cavity and suggest novel research directions for targeting this critical research question.

Bacterial adhesion and biofilm formation on yttria-stabilized, tetragonal zirconia and titanium oral implant materials with low surface roughness - an in situ study.

Bacterially-driven mucosal inflammation and the development of periimplantitis can lead to oral implant failure. In this study, initial bacterial adhesion after 2 h, and biofilm formation after 1 day and 3 days, were analysed in situ on novel 3 mol % yttria-stabilized tetragonal zirconia polycrystal samples, as well as on alumina and niobium co-doped yttria-stabilized tetragonal zirconia samples. Pure titanium implant material and bovine enamel slabs served as controls. The initially adherent oral bacteria were determined by 4',6-diamidino-2-phenylindole-staining. Biofilm thickness, surface covering grade and content of oral streptococci within the biofilm were measured by fluorescence in situ hybridization. No significant differences between the ceramic and titanium surfaces were detectable for either initial bacterial adhesion or the oral streptococci content of the in situ biofilm. The oral biofilm thickness on the implant surfaces were almost doubled after three days compared to the first day of oral exposure. Nevertheless, the biofilm thickness values among the different implant surfaces and controls did not differ significantly for any time point of measurement after 1 day or 3 days of biofilm formation. Significant differences in the covering grade were only detected between day 1 and day 3 for each tested implant material group. The content of oral streptococci increased significantly in parallel with the increase in biofilm age from day 1 to day 3. In conclusion, oral implant zirconia surfaces with low surface roughness are comparable to titanium surfaces with respect to initial bacterial adhesion and biofilm formation.

Peri-implant bone response to retrieved human zirconia oral implants after a 4-year loading period: A histologic and histomorphometric evaluation of 22 cases.

AIM: To evaluate the bone tissue response to surface modified zirconia oral implants retrieved from humans. MATERIALS AND METHODS: Twenty-nine one-piece zirconia implants showed increased marginal bone loss and did not response to the applied peri-implantitis therapy. After their removal using a trephine bur, 22 of the implant-bone biopsies were suitable for an evaluation and immediately immersed in formalin for two weeks. Subsequent, the retrieved specimens were histologically prepared and the regions still showing osseointegration computer-assisted analyzed regarding the bone-to-implant contact (BIC) and bone density using a transmitted-light microscope. RESULTS: The removed implants were in situ for a mean time period of 47.7 months. After their removal, compact bone could be depicted at the apical regions. The remaining bone that was attached to the implants contained a regular lamellar structure with osteons and osteocytes. The BIC ranged from 58.1% to 93.7% (mean: 76.5%) and the bone area/density within the implant threads ranged from 57% to 97.2% (mean: 84.8%). CONCLUSIONS: The porous zirconia implants showed a sufficient BIC in the areas where bone still was attached. Although the implants had to be removed due to increased bone loss, it seems that the presented zirconia implant surface per se elicited appropriate osseointegration. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1622-1631, 2016.

Evaluation of alumina toughened zirconia implants with a sintered, moderately rough surface: An experiment in the rat.

OBJECTIVE: Alumina toughened zirconia (ATZ) is more fracture resistant than unmodified zirconia and has been shown to be a viable substrate for the growth of osteoblasts. In this study, we examined the histological and biomechanical behavior of moderately roughened ATZ implants in rat femoral bone. METHODS: Miniature implants made of ATZ with pore-building polymers sintered onto the surface and electrochemically anodized titanium (TiUnite®) were placed into the femurs of Sprague-Dawley rats. Implant surface topography was analyzed by 3D laserscan measurements and scanning electron microscopy (SEM). After a healing period of 14 and 28 days, respectively, histologic and biomechanical testing was performed. RESULTS: Under the SEM, the TiUnite® surface could be clearly distinguished from the ATZ surface, but 3D laserscan measurements indicated a moderately rough surface topography for both, TiUnite® (Sa=1.31µm) and ATZ (Sa=1.51µm). The mean mineralized bone-to-implant contact showed the highest values after 14 and 28 days for TiUnite® (58%/75%) as compared to ATZ (24%/41%). The push-in values after a healing period of 14 and 28 days, respectively, increased from 20N to 39N for TiUnite® and from 10N to 25N for ATZ. SIGNIFICANCE: Our findings suggest that the moderately roughened ATZ implant surface is well accepted by rat bone tissue. However, compared to titanium, the osseointegration-process of ATZ seems to proceed more slowly in that early phase of implant integration.

The Effect of UV Treatment on the Osteoconductive Capacity of Zirconia-Based Materials.

OBJECTIVE: Improvements in the bioactivity of zirconia implants for accelerated healing and reduced morbidity have been of continuing interest in the fields of dentistry and orthopedic surgery. The aim of the present study was to examine whether UV treatment increases the osteoconductivity of zirconia-based materials. MATERIALS AND METHODS: Smooth and rough zirconia-based disks and cylindrical implants were treated with UV light for 15 min and subsequently placed in rat femurs. Surface characterization was performed using scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle measurements. RESULTS: In vivo histomorphometry revealed that the percentage of bone-implant contact and the amount of bone volume, formed around UV-treated implants, increased by 3-7-fold for smooth surfaces and by 1.4-1.7-fold for rough surfaces compared to non-treated specimens at Weeks 2 and 4 of healing, respectively. A biomechanical test showed that UV treatment accelerated the establishment of bone-zirconia integration and enhanced the strength of the bone-implant interface by two-fold. Additionally, surface characterization of the zirconia disks revealed that UV treatment decreased the amount of surface carbon and converted the hydrophilic status from hydrophobic to superhydrophilic. CONCLUSIONS: This study indicates that UV light pretreatment enhances the osteoconductive capacity of zirconia-based materials.

Effect of two different healing times on the mineralization of newly formed bone using a bovine bone substitute in sinus floor augmentation: a randomized, controlled, clinical and histological investigation.

PURPOSE: To investigate the amount of the mineralization of a bovine bone substitute material in sinus floor augmentation after healing times of 3 and 6 months. MATERIALS AND METHODS: Fifty-one patients were randomized into two healing time groups and received sinus floor augmentations with a bovine bone material. After 3 or 6 months of healing, trephine bone biopsies were retrieved. The biopsies were processed for histological and histomorphometric evaluations to primarily investigate the amount of mineralized bone in the augmented area and secondarily compare the amount of mineralized bone in the augmented area and in the pristine bone. Statistical tests were performed to analyse the fraction of the mineralized bone (p < 0.05). RESULTS: The biopsies of both groups showed remnants of the well-integrated bone substitute material. The histology revealed osteoblasts, osteocytes with osteoid, and osteoclasts. The mean percentage of mineralized bone in the augmented area was 23.8% (3 months group) and 23.6% (6 months group; p = 0.9246); the amount of remaining bone substitute material was 35% (3 months group) and 33.9% (6 months group; p = 0.6325). CONCLUSION: It can be concluded that the bone maturation in the augmented sinus using the bovine bone material is similar after 3 and 6 months. Thus, implant installation after 3 months following a lateral window sinus floor augmentation approach using a bovine bone material seems to be clinically acceptable.

Evaluation of Guided Bone Regeneration around Oral Implants over Different Healing Times Using Two Different Bovine Bone Materials: A Randomized, Controlled Clinical and Histological Investigation.

PURPOSE: To evaluate the potential of two bone substitute materials and the influence of different healing periods in guided bone regeneration therapy of osseous defects around implants. MATERIALS AND METHODS: Twenty-four edentulous patients received implants in the region of the lost lower incisors. Around two standardized osseous defects were created, treated either with a 50:50 mixture of PepGen P-15® and OsteoGraf®/N-700 (test group) or with BioOss® (control group), and covered with titanium membranes. After healing periods of 2, 4, 6, or 9 months, the implants were removed together with the surrounding bone and subsequently prepared for histological evaluations. RESULTS: Defect depths in both groups showed a clinical reduction after intervention. The histologically measured distance from the implant shoulder to the first point of bone-implant contact (BIC) after treatment did not differ between the two groups. The healing time influenced the level of the first point of BIC, with a longer healing period producing a more coronal first point of BIC. A greater percentage BIC and a higher fraction of mineralized bone were found in the pristine bone area compared with the augmented defect area. CONCLUSION: It can be concluded that in the treatment of osseous defects around oral implants, both materials were equally effective bone substitute materials when used in combination with guided bone regeneration.

Osteoblast and bone tissue response to surface modified zirconia and titanium implant materials.

OBJECTIVE: This study examined the in vitro and in vivo response of osteoblasts to a novel, acid-etched and sandblasted zirconia surface. METHODS: Osteoblastic hFOB 1.19 cells were cultured either on electrochemically anodized titanium (TiUnite(®)), machined titanium (Ti-m), sandblasted and acid-etched zirconia (TZP-proc), and machined zirconia (TZP-A-m). The surface topography of the various substrates was analyzed by 3D laserscan measurements and scanning electron microscopy. At culture days 1, 3, 7, 14, 21, and 28, cell proliferation was determined. Gene expression was analyzed using RT-PCR. Histologic analysis and biomechanical testing was performed on miniature implants placed in the rat femur. RESULTS: During the first 7 days, a retarded cell proliferation was observed on the TiUnite(®) surface. After 28 days of cultivation, cell proliferation reached similar levels on all surfaces. An up-regulation of bone and extracellular matrix specific genes could be seen for TZP-proc at day 21. The mean bone-implant contact rate after a healing period of 14 and 28 days, respectively, was higher for TiUnite(®) than for TZP-proc. At 28 day, the biomechanical test showed significantly higher values for TiUnite(®) than for all other surfaces. SIGNIFICANCE: The novel, rough zirconia surface was accepted by hFOB 1.19 cells and integrates into rat bone tissue. However, osseointegration seemed to proceed more slowly and to a lesser extent compared to a moderately roughened titanium surface.

Sinus augmentation with bovine hydroxyapatite/synthetic peptide in a sodium hyaluronate carrier (PepGen P-15 Putty): a clinical investigation of different healing times.

PURPOSE: Augmentation of the sinus floor with autogenous bone often requires an extra donor site, which is associated with a risk of morbidity, and current grafting protocols involve healing times of up to 9 months. In this prospective in vivo study, the time-dependent efficacy of PepGen P-15 Putty, a combination of bovine hydroxyapatite and synthetic peptide in a sodium hyaluronate carrier, was evaluated in sinus grafting. MATERIALS AND METHODS: Twenty-four edentulous patients received bilateral sinus augmentations with PepGen P-15 Putty, which mimics the cell-binding domain of type I collagen responsible for cell migration, differentiation, and proliferation. The patients were randomly divided into four groups of six patients each, corresponding to 2, 4, 6, and 9 months of healing postaugmentation. After these time intervals, bone biopsy specimens were retrieved through the alveolar bone crest into the augmented sinus, and subsequently oral implants were placed. The specimens were immersed in buffered formalin, scanned with a desktop microcomputed tomography machine, and processed for histologic and histomorphometric evaluation. A mixed model was used for statistical analysis. RESULTS: Three-dimensional microcomputed tomography depicted the distinct structure of trabecular bone encompassing remnants of PepGen, and histologic evaluation revealed osteoblasts and osteoid with osteocytes in the vicinity of the PepGen particles at all healing stages. Histomorphometric results indicated an increase in the newly formed bone fraction in the specimens as follows: 21.3% (± 2.33) at 2 months, 21.9% (± 8.9) at 4 months, 28.5% (± 6.9) at 6 months, and 29.8% (± 11.8) at 9 months. The differences were statistically insignificant. All implants placed in the augmented sites integrated and were restored prosthetically. CONCLUSIONS: PepGen can be used successfully for maxillary sinus augmentation. These data provide evidence that implant placement, even after only 2 months of healing, may be possible.

Biomechanical and histological behavior of zirconia implants: an experiment in the rat.

OBJECTIVE: This study aimed at evaluating the integration of zirconia implants in a rat femur model. MATERIAL AND METHODS: Zirconia implants with two distinct surface topographies were compared with titanium implants with similar topographies. Titanium and zirconia implants were placed into the femurs of 42 male Sprague-Dawley rats. Four groups of implants were utilized: machined zirconia implants, zirconia implants with a rough surface, machined titanium implants, and titanium implants with an electrochemically roughened surface. After a healing period of 28 days, the load-bearing capacity between the bone and the implant surface was evaluated by a push-in test. Additionally, after a healing period of 14 and 28 days, respectively, bone tissue specimens containing the implants were processed and histologically analyzed. RESULTS: The mean mineralized bone-to-implant contact showed the highest values after 14 and 28 days for the rough surfaces (titanium: 36%/45%; zirconia: 45%/59%). Also, the push-in test showed higher values for the textured implant surfaces, with no statistical significance between titanium (34 N) and zirconia (45.8 N). CONCLUSIONS: Within the limits of the animal investigation presented, it was concluded that all tested zirconia and titanium implant surfaces were biocompatible and osseoconductive. The presented surface modification of zirconia implants showed no difference regarding the histological and biomechanical results compared with an established electrochemically modified titanium implant surface.

The gene-expression and phenotypic response of hFOB 1.19 osteoblasts to surface-modified titanium and zirconia.

The osteoblastic cell-line hFOB 1.19 with the potential to proliferate and differentiate revealed that cellular differentiation is not affected by material and roughness on newly developed zirconia implant materials. Materials under investigation were surfaces machined titanium (Ti-m), modified titanium (TiUnite, machined zirconia (TZP-A-m), modified zirconia (ZiUnitemachined alumina-toughened zirconia (ATZ-m) and modified alumina-toughened zirconia (ATZ-mod). After surface description by scanning electron microscopy (SEM) and atomic force microscopy (AFM), cellular proliferation (EZ4U, Casy1) and differentiation were examined after days 1, 3, 7, 14, 21, and 28. Osteogenic differentiation was visualized by alkaline phosphatase staining, mineralization assay (alizarin red) and by expression analysis (RT-PCR) of bone- and extracellular matrix-related genes. Proliferation on rough surfaces was reduced on both titanium and zirconia. Cell-attachment and cytoskeleton organization documented by confocal laser scanning microscopy (CLSM) elucidated attenuated cell attachment within the first 4h to be the reason for impaired proliferation. A specific up-regulation of m-RNAs in an early event (RUNX2, NELL-1, RUNX3, and BMP7) and a late event (Integrin B3) could be observed on TiUnite and ZiUnite. For titanium an up-regulation of IBSP and Integrin B1 could be described at day 21. In total, differentiation was neither affected by material nor by roughness.

Initial osteoblast-like cell response to pure titanium and zirconia/alumina ceramics.

OBJECTIVES: Outstanding mechanical properties, resistance to scratching and high biocompatibility make zirconia/alumina ceramics interesting for dental applications. To solve the problem of the well-known low temperature degradation and to provide stable mechanical properties a novel zirconia alloy ((Y,Nb)-TZP/alumina) was developed. The aim of this study was to investigate the initial bone cell response to this new zirconia/alumina composite ceramic. METHODS: HOS cells were cultured on zirconia/alumina composite (Zc) and pure titanium (Ti) discs. Surface topography was examined by atomic force microscopy (AFM), cell morphology by scanning electron microscopy (SEM). Cell proliferation (MTS) and alkaline phosphatase activity was measured at 1, 4 and 8 days. The mRNA expression of Cycline D1, the cell cycle regulating gene, integrin beta 1, osteonectin (ON) and beta-actin were evaluated by RT-PCR analysis after 12, 24 and 48 h. RESULTS: Both substrates showed a very smooth character with R(a)-values in the range of 0.002-0.113 microm supporting a continuous cellular growth. After 8 days, cell proliferation on Zc was higher than on Ti. The mRNA expression of cyclin D1 showed similar activity after 48 h on both surfaces, ALP activity was higher on Zc after 8 days. ON expression however showed no difference between the two groups. SIGNIFICANCE: Our data demonstrate that this new zirconia composite ceramic showed at least equivalent or slightly better biological response of osteoblast-like HOS cells than pure titanium during a short-time cell culture period.

Behavior of CAL72 osteoblast-like cells cultured on zirconia ceramics with different surface topographies.

OBJECTIVES: Because of its inherent strength, biocompatibility, and tooth-like color, zirconia ceramics have the potential to become an alternative to titanium as dental implant material. This study aimed at investigating the osteoblastic response to yttrium-stabilized tetragonal zirconia polycrystal (Y-TZP) with different surface topographies. METHODS: CAL72 osteoblast-like cells were cultured on machined (TZP-m), airborne particle abraded (TZP-s), and airborne particle abraded and acid-etched Y-TZP (TZP-sa) surfaces. Polystyrene and airborne particle abraded with large grit and acid-etched (SLA) titanium served as a reference control. The surface topography was examined by scanning electron microscopy (SEM) and profilometry. At culture days 3, 6, and 12, cell proliferation, at day 12 cell morphology, and cell-covered surface area were determined. RESULTS: The surface roughness of Y-TZP was increased by airborne particle abrasion and additionally by acid etching. No statistically significant differences were found between average roughness (R(a)) and maximum peak-to-valley height (R(p-v)) values of airborne particle abraded and acid-etched Y-TZP and SLA titanium. Whereas the cell proliferation assay revealed statistically significant greater values at day 3 for surface-treated Y-TZP and polystyrene cultures as compared with machined Y-TZP, no differences between the Y-TZP groups, SLA titanium, and polystyrene were observed at culture days 6 and 12. CONCLUSIONS: Cell morphology and cell-covered surface area were not affected by the type of substrate. The results suggest that roughened Y-TZP is an appropriate substrate for the proliferation and spreading of osteoblastic cells.

On-line analysis of CAL72 cells on two different titanium surfaces in a perfusion micro-bioreactor.

OBJECTIVES: The aim of the present experiment was to test a prototype microsensoric measuring system (micro-bioreactor) for the investigation of the biocompatibility of different titanium surfaces in a cell culture model. METHODS: Osteosarcoma cells of the cell line CAL72 were seeded onto titanium plates (10mm x 10mm x 1mm) and inserted into the culture chamber of the micro-bioreactor. Titanium plates with two different surface topographies (machined and titanium plasma-sprayed [TPS]) were used for this pilot investigation. Plastic plates served as controls. The online-sensoric device of the micro-bioreactor allowed the continuous monitoring of the metabolism of the cells and the control of the culture conditions. Over a period of 17 h changes in O(2)-consumption in the medium were measured by micro-electrodes and registered by the software of the system. The metabolic activity of the cells was calculated from the difference between the bypass and the chamber values. The cell proliferation and vitality were analyzed before and after the perfusion time in the micro-bioreactor. The cell morphology was studied using scanning electron microscopy. RESULTS: The cells on the machined surfaces showed the highest oxygen consumption after 15 h, after that it decreased. The cells on the TPS plates showed a lower oxygen consumption, which remained stable after 17 h. The highest oxygen consumption was seen with the cells on the control plastic plates. Concerning cell proliferation analysis, it could be shown that more vital CAL72 cells seeded onto TPS and plastic could be detected after the passage through the micro-bioreactor. Hence, the number of vital cells on the machined surface was reduced after the passage. SIGNIFICANCE: Within the limits of this experiment, the presented micro-bioreactor system could offer a valuable method to examine the dynamic interactions of cells and materials under defined in vitro experimental conditions. While the presented system is already successfully used in the ecological/ecotoxicological field, its routine use for investigating dental materials on a cellular level has to be evaluated.

A systematic review of the influence of different titanium surfaces on proliferation, differentiation and protein synthesis of osteoblast-like MG63 cells.

OBJECTIVES: Titanium is the standard material for dental and orthopaedical implants. The good biocompatibility has been proven in many experimental and clinical investigations. Different titanium topographies were tested in vitro using different cell culture models. The aim of this systematic review was to evaluate and summarize the medical/dental literature to assess on which kind of titanium surface structure the osteoblast-like osteosarcoma cells MG63 show the best proliferation and differentiation rate, and the best protein synthesis. METHODS: A systematic search was carried out using different on-line databases (PubMed, Web of Science, Cochrane Library, International Poster Journal), supplemented by handsearch in selected journals and by examination of the bibliographies of the identified articles. Inclusion and exclusion criterias were applied when considering relevant articles. Studies which met the inclusion criteria were included and data extraction was undertaken by one reviewer. RESULTS: The search yielded 348 references. Nine articles referring to nine different studies were relevant to our question. Additionally 8 less relevant articles were identified. It was found that regularly textured surfaces of pure titanium with R(a) values (average roughness) of around 4 mum are well-accepted by MG63 cells. CONCLUSIONS: The surfaces and culture conditions vary widely. Therefore it is still difficult to recommend one particular surface. It seems that there are no differences in cell proliferation and differentiation on surfaces treated by blasting and etching. Standardization in fabrication and size of the different test surfaces as well as homogeneity in culture times and plating densities should be aspects for future research.

Loaded custom-made zirconia and titanium implants show similar osseointegration: an animal experiment.

BACKGROUND: Zirconia might be an alternative material to titanium for dental implant fabrication. The aim of the present study was to investigate the histological behavior (osseointegration) of loaded zirconia implants in an animal model and to compare it with the behavior of titanium implants. METHODS: Five months after extraction of the upper anterior teeth, 12 custom-made titanium implants (control group) and 12 custom-made zirconia implants (test group) were inserted in the extraction sites in six monkeys. Before insertion, the titanium implant surfaces were sandblasted with Al2O3 and subsequently acid-etched. The zirconia implants were only sandblasted. Six months following implant insertion, impressions were taken for the fabrication of single crowns. A further 3 months later, nonprecious metal crowns were inserted. Five months after insertion of the crowns, the implants with the surrounding hard and soft tissues were harvested, histologically prepared, and evaluated under the light microscope regarding the peri-implant soft tissue dimensions and mineralized bone-to-implant contact. RESULTS: No implant was lost during the investigational period. The mean height of the soft peri-implant tissue cuff was 5 mm around the titanium implants and 4.5 mm around the zirconia implants. No statistically significant differences were found in the extent of the different soft tissue compartments. The mean mineralized bone-to-implant contact after 9 months of healing and 5 months of loading amounted to 72.9% (SD: 14%) for the titanium implants and to 67.4% (SD: 17%) for the zirconia implants. There was no statistically significant difference between the different implant materials. CONCLUSION: Within the limits of this animal experiment, it can be concluded that the custom-made zirconia implants osseointegrated to the same extent as custom-made titanium control implants and show the same peri-implant soft tissue dimensions.

Hard tissue reaction to dual acid-etched titanium implants: influence of plaque accumulation. A histological study in humans.

OBJECTIVES: The aim of this investigation was to evaluate histologically, histometrically, and histomorphometrically the influence of plaque accumulation on the peri-implant hard tissues. MATERIAL AND METHODS: Twelve fully edentulous subjects were selected for this investigation. Four to five standard titanium screw implants were placed interforaminally. Two small custom-made screw implants were incorporated in the region of the former first molar, one in each lower quadrant. One month after abutment connection, plaque control was terminated randomly at one of the custom-made implants and continued at the other implant. The custom-made implants and the surrounding tissue were harvested after different time points of plaque accumulation (7, 21, or 90 days). Thus, according to the plaque control program and implant removal time, there were six groups each with four implants for investigation. After histologic processing of the biopsies, histologic, histometric, and histomorphometric analyses were performed. RESULTS: Four of the 24 implants were unavailable for analysis. One implant was mobile at abutment connection and another implant was mobile 2 weeks after abutment connection; both implants had to be removed. The bone around two implants was destroyed during the removal with the trephine bur; therefore, these two implants could not be evaluated histologically either. No differences in the histologic appearance of the peri-implant bone between the different groups could be observed. Histomorphometrically, the implant surface in contact with mineralized bone as a fraction of the implant surface (measured from the buccal to the lingual implant shoulder) varied between 59% and 73%. The implant surface in contact with mineralized bone from the first bone-to-implant contact buccally to the first bone-to-implant contact lingually varied between 80.6% and 91.5%. The values for the distance from the implant shoulder to the first bone-to-implant contact, i.e. peri-implant bone loss, varied from 1.1 to 2.2 mm. No statistically significant differences could be found between any of the evaluated variables. CONCLUSIONS: This clinical investigation is the first to evaluate the hard tissue reactions to different plaque accumulation periods. Within the limits of this experiment, it can be concluded that the bone reaction toward the different plaque accumulation periods and in the different plaque control/accumulation groups were similar. It can be further concluded that the observed bone loss is rather attributed to the establishment of the biologic width than to plaque accumulation.