Soft tissue integration versusearly biofilm formation on different dental implant materials

Abstract

Objective

Dental implants anchor in bone through a tight fit and osseo-integratable properties of the implant surfaces, while a protective soft tissue seal around the implants neck is needed to prevent bacterial destruction of the bone-implant interface. This tissue seal needs to form in the unsterile, oral environment. We aim to identify surface properties of dental implant materials (titanium, titanium-zirconium alloy and zirconium-oxides) that determine the outcome of this “race-for-the-surface” between human-gingival-fibroblasts and different supra-gingival bacterial strains.

Methods

Biofilms of three streptococcal species or a Staphylococcus aureus strain were grown in mono-cultures on the different implant materials in a parallel-plate-flow-chamber and their biovolume evaluated using confocal-scanning-laser-microscopy. Similarly, adhesion, spreading and growth of human-gingival-fibroblasts were evaluated. Co-culture experiments with bacteria and human-gingival-fibroblasts were carried out to evaluate tissue interaction with bacterially contaminated implant surfaces. Implant surfaces were characterized by their hydrophobicity, roughness and elemental composition.

Results

Biofilm formation occurred on all implant materials, and neither roughness nor hydrophobicity had a decisive influence on biofilm formation. Zirconium-oxide attracted most biofilm. All implant materials were covered by human-gingival-fibroblasts for 80–90% of their surface areas. Human-gingival-fibroblasts lost the race-for-the-surface against all bacterial strains on nearly all implant materials, except on the smoothest titanium variants.

Significance

Smooth titanium implant surfaces provide the best opportunities for a soft tissue seal to form on bacterially contaminated implant surfaces. This conclusion could only be reached in co-culture studies and coincides with the results from the few clinical studies carried out to this end.

Introduction

The application of dental implants, directly anchored in the bone, can be considered as one of the major advances in modern dentistry and provides considerably more comfort for patients in the restoration of function after tooth loss than dentures or bridges. Up to now, pure titanium has been the most widespread and successfully used material for these implants because it is biocompatible, corrosion resistant, lightweight and durable, while it can be easily prepared in many different shapes and textures without affecting its biocompatibility . The fate of a biomaterial implant can be envisaged as a race for the surface between bacteria and host tissue cells . If bacteria win the race for the surface, the implant is mostly lost and has to be replaced, as bacteria adhere to an implant surface in their protective, biofilm mode of growth . On the other hand, if tissue cells are able to integrate the implant surface, this yields the best available protection against infection . Considering the relatively unsterile conditions under which dental implants are inserted and have to osseo-integrate, one can wonder how the race for the surface can ever be won by host tissue cells on a dental implant surface. Yet, early failure of dental implants occurs in “only” 8% and 5% of the implants in the maxilla and the mandible, respectively .

Success or failure of dental implants is directly related to the degree of integration of the implant material by surrounding soft and hard tissues versus biofilm formation . Osseo-integration is nowadays less of a problem than the formation of a soft tissue seal and is achieved by a tight fit of the implant in the bone and an osseo-integrative morphology and/or chemistry of the implant surface. Non-osteogenic soft tissue integration of the neck of the implant surface is necessary to create a biological seal that protects against invasion of periodontopathogens toward the bone . Surface roughness and hydrophobicity, as a corollary of the chemical composition of the implant surface, are generally considered as the key-properties of dental implant surfaces for both tissue integration and biofilm formation .

Early failure of dental implants is initiated by adhesion of streptococci and other initial colonizers preventing the formation of a soft tissue seal. Recently Staphylococcus aureus has also been identified as an initial colonizer of dental implants . Late peri-implantitis constitutes a totally different race for the surface, where periodontopathogens, other than supra-gingival colonizers, have to displace periodontal ligament fibroblasts from the neck of the implant surface , ultimately causing bone loss and possible loss of an entire implant.

The race for the surface between human gingival fibroblasts (HGF) cells and initial colonizers seems like an impossible one to win for HGF cells in the human oral cavity, as it is loaded with different microbial strains and species. Although dental implant surface properties have been investigated with respect to either biofilm formation or tissue interaction in mono-culture studies (encompassing either bacteria or tissue cells), co-culture experiments in which bacteria and tissue cells are actually competing in one and the same experiment for an implant surface have never been carried out on dental implant surfaces.

The aim of this study was to compare the outcome of the race for the surface in co-culture experiments between contaminating supra-gingival bacterial strains and HGF cells on different, currently used dental implant materials in an early failure model, used earlier to study the race for the surface on orthopedic implant materials . Such a study is direly needed, as benefits of different titanium (Ti) surface modifications, as well as of the use of titanium-zirconium (TiZr) alloys and zirconia (ZrO 2 ), have only been evaluated in mono-culture studies with little clinical relevance or in clinical studies with highly limited statistical power . The bacterial strains involved in the current study include supra-gingival strains ( Streptococcus oralis J22, Streptococcus mitis BMS, Streptococcus salivarius HB and S. aureus ATCC 25923), as involved in early failure of dental implants.

Materials and methods

Implant materials

All implant materials used in this study were provided by Institut Straumann AG (Basel, Switzerland) and were received as discs (diameter 5 mm, thickness 1 mm). Three different implant materials were included in this study, comprising titanium (Ti, cold-worked, grade 4), titanium-zirconium alloy (TiZr, 15% (wt) Zr) and zirconium oxide (ZrO 2 , Y-TZP). The materials were modified according to different procedures, indicated as

  • P: mirror-polished with finally a 0.04 μm SiO 2 suspension and cleaned by Deconex solution, followed by concentrated nitric acid and water.

  • M: ground to mimic the machined part of the implant and washed with Deconex solution, followed by concentrated nitric acid and water.

  • MA: ground and then acid-etched with a boiling mixture of concentrated HCl and H 2 SO 4 (or hot hydrofluoric acid in case of ZrO 2 ) and rinsed with concentrated nitric acid and water.

  • modMA: ground and acid-etched with HCl/H 2 SO 4 , as described above, but rinsed with water only under N 2 protection and directly stored individually in glass containers, filled with an isotonic NaCl solution, protected by N 2 filling.

M, MA, modMA and P modifications were all applied to Ti, whereas TiZr alloy and ZrO 2 were only modified according to M and modMA (TiZr) or MA (ZrO 2 ). M, MA and P modified discs were individually packed in aluminum foil and sealed in plastic.

All discs were sterilized in their respective packagings by γ-irradiation at 25–42 kGy.

Implant surface characterization

The hydrophobicity of the implant surfaces was determined by water contact angle measurements at room temperature with a home-made contour monitor, using the sessile drop (1–1.5 μl droplets) method. All values reported are averages over three different implant surfaces, while five droplets were placed on each disk.

The elemental surface composition of the implant surfaces was measured using an S-probe X-ray photoelectron spectrometer (Surface Science Instruments, Mountain View, CA, USA), equipped with an aluminum anode (10 kV, 22 mA) and a quartz monochromator. The direction of the photoelectron collection angle was 55 degrees with the sample surface and the electron flood gun was set at 10 eV. Survey scans were made with a 1000 μm × 250 μm spot and pass energy of 150 eV and used to calculate elemental surface compositions of the implant surfaces. The O 1s photoelectron binding energy peak was also recorded with a pass energy of 50 eV and decomposed into a component at 530.1 eV due to oxygen involved in oxide bonds and other components due to, for instance oxygen involved in carbonaceous contamination. All experiments were done on two discs of each implant material, while two separate measurements were taken on each sample.

The average surface roughness of the different implant surfaces was assessed with an atomic force microscope (Nanoscope IIIa Dimension™3100, Bruker, Santa Barbara, CA, USA), operated in the contact mode and using a Si 3 N 4 cantilever tip (DNP from Veeco, Woodbury, NY, USA) with a spring constant of 0.06 N/m. Analyses were done on three randomly selected places per sample disk, employing three discs for each implant material. The surface morphology of different implant surfaces was investigated using scanning electron microscopy (SEM). The specimens were fixed on SEM-stub-holders and visualized in a field emission scanning electron microscope (FE-SEM type 6301F, JEOL Ltd., Tokyo, Japan) at 2 kV and a magnification of 3000×.

Bacterial strains and culturing

For biofilm formation, S. oralis J22, S. mitis BMS, S. salivarius HB, and S. aureus ATCC 25923 were used. Strains were streaked onto blood agar plates and incubated for 24 h at 37 °C. Then, a fresh colony was inoculated in 10 ml of appropriate growth medium. S. oralis J22, S. mitis BMS, S. salivarius HB were inoculated in Todd Hewitt Broth (THB; OXOID, Basingstoke, England) and S. aureus ATCC 25923 was inoculated in Tryptone Soya Broth (TSB; OXOID, Basingstoke, England) for 24 h at 37 °C. Subsequently, 10 ml of the bacterial culture was inoculated into 200 ml growth medium and a second culture was grown for 16 h at 37 °C. Bacteria were harvested by centrifugation at 4000× g for 5 min at 10 °C, and streptococci washed twice with sterile adhesion buffer (2 mM potassium phosphate, 50 mM potassium chloride, and 1 mM calcium chloride, pH 6.8) while S. aureus ATCC 25923 was washed in sterile phosphate buffered saline (PBS, 10 mM potassium phosphate, 0.15 M NaCl, pH 7.0). Subsequently, bacteria were sonicated on ice (3 × 10 s) in sterile buffer at 30 W (Vibra Cell model 375; Sonics and Materials, Danbury, CT, USA) in order to break bacterial aggregates. Bacteria were counted in a Bürker–Türk counting chamber and the bacterial suspension was further diluted to a concentration of 3 × 10 5 bacteria per ml for all experiments.

Bacterial adhesion and biofilm formation

Bacterial adhesion and biofilm formation were studied on the bottom plate of a parallel plate flow chamber (175 mm × 17 mm × 0.75 mm). The bottom plate consisted of transparent poly (methyl methacrylate) in which six 1 mm deep inserts were prepared in the center region of the plate that could each hold one implant material disk (see Fig. 1 ). In this way, six different implant materials could be analyzed in one experiment, therewith reducing influence of biological variations between bacterial cultures. Poly (methyl methacrylate) bottom plates were cleaned in a 2% RBS detergent solution (Omniclean, Breda, The Netherlands) under sonication and thoroughly rinsed in demineralized water, 70% ethanol, water again and finally washed with sterile ultrapure water prior to use. The flow chamber used was equipped with heating elements and kept at 37 °C throughout an experiment. Bacterial deposition was observed with a CCD camera (Basler AG, Germany) mounted on a phase-contrast microscope Leica DM2000 (Leica Microsystems Ltd., Germany) with a 40× objective lens.

Fig. 1
Schematics of the parallel plate flow chamber employed, with the inserts for different samples in the bottom plate indicated.

Prior to each experiment, all tubes and the flow chamber were filled with suitable buffer. After removing all air bubbles from the system, a bacterial suspension in buffer was perfused through the system at a shear rate of 11 s −1 . Images were obtained as a function of time.

In a separate set of experiments using fluorescence microscopy after staining of adhering bacteria, it was first established that all materials including the poly (methyl methacrylate) bottom plate, initially attracted identical numbers of adhering bacteria. Hence, during final experiments phase-contrast microscopic enumeration of the number of bacteria on the transparent bottom plate could be used to control the number of initially adhering bacteria on the different implant surfaces.

As soon as the number of adhering bacteria amounted 10 3 cm −2 , flow was switched to buffer again in order to remove un-adhering bacteria from the tubes and chamber. Subsequently, modified culture medium (Dulbecco’s modified Eagle’s medium high glucose with 25 mM HEPES supplemented with 10% (v/v) fetal calf serum, 0.2 mM ascorbic acid-2-phosphate and 2% (v/v) of the appropriate bacterial growth medium, (for details see section below “Adhesion, spreading and growth of human gingival fibroblasts”) was perfused through the system at a low shear rate of 1.67 s −1 for 24 h, in order to allow the adhering bacteria to grow into a biofilm.

After 24 h of growth, biofilms were stained with a vitality staining solution, containing 3.34 mM SYTO 9 and 20 mM propidium iodide (Molecular Probes Inc, USA) in PBS. Staining was done in the flow chamber for 15 min in the dark at room temperature. Next biofilms were examined with a confocal laser scanning microscope (CLSM, Leica DMRXE with a confocal TCS SP2 unit) with a water objective (HCX APO L 40.0 × 0.80 W) using 488 nm excitation and emission filters of 500–550 nm and 605–720 nm to reveal live or dead bacteria. Images were taken over the depth of a biofilm in sequential steps of 0.8 μm. Subsequently, the stacks of images acquired were analyzed for the total biofilm volume per unit area with the program “COMSTAT” .

Human gingival fibroblasts and culturing

Gingival fibroblasts were obtained from the American Type Culture Collection (HGF-1, ATCC-CRL-2014). Cells were routinely grown in monolayer cultures in Dulbecco’s modification or Eagle’s medium (DMEM) containing 25 mM HEPES and further supplemented with 10% (v/v) Fetal Bovine Serum, 0.2 mM ascorbic acid-2-phosphate at 37 °C in a humidified atmosphere with 5% CO 2 . At 70–80% confluence the fibroblast cultured were passaged using a Trypsin-EDTA solution (Invitrogen, Breda, The Netherlands). Thus grown cells, between passage 4–10 were used in all experiments, i.e. mono- and co-culture studies, pursuing further growth in modified culture medium in order to grow both oral supra-gingival bacteria and HGF cells simultaneously in the same growth medium.

To develop a modified culture medium allowing growth of HGFs and bacteria, bacterial medium (THB or TSB) and DMEM were combined in different ratios and growth of both bacteria and HGF cells examined. To examine HGF growth in modified culture media with different amounts of THB or TSB added, 200,000 cells, were suspended in modified culture media and seeded into T25 cell culture flasks. After incubation at 37 °C in a humidified 5% CO 2 atmosphere for 48 h, cells were immuno-stained and their spreading and morphology was assessed from micrographs, obtained using fluorescence microscopy. The bacterial strains were separately inoculated from agar plates in 10 ml THB or TSB for 24 h. These cultures were used to inoculate 10 3 bacteria in second 10 ml cultures of modified culture media which were incubated for 24 h (“pre-cultures”). From these pre-cultures, 0.5 ml was used to inoculate 9.5 ml of modified culture medium and grown overnight. Bacteria were counted using a Bürker–Türk counting chamber. Based on the combined results, modified culture medium comprised of DMEM with 2% THB (for S. oralis , S. mitis , S. salivarius ) or TSB (for S. aureus ) added, was chosen for further studies and will be denoted as “modified culture medium” in the remainder of this study.

Adhesion, spreading and growth of human gingival fibroblasts

Discs of each implant material were placed in 48-wells plates and 1 ml of HGF cell suspension in modified culture medium with a concentration of 20,000 cells/ml was added. Cells were maintained at 37 °C in a humidified 5% CO 2 for 48 h. After 48 h, HGF cells were fixed in 3.7% paraformaldehyde in cytoskeleton stabilization buffer (0.1 M Pipes, 1 mM EGTA, 4% (w/v) polyethylene glycol 8000, pH 6.9). After 5 min, the fixation solution was replaced by fresh 3.7% paraformaldehyde for another 10 min. Subsequently cells were incubated in 0.5% Triton X-100 for 3 min, and stained for 30 min in 5 ml PBS with 1% albumin containing 0.4 μg/ml DAPI and 2 μg/ml of TRITC-phalloidin. Adhering cells were subsequently washed in PBS with 1% bovine serum albumin for 5 min, washed in PBS, stored in PBS and examined with Leica DMIL fluorescence microscope (Leica Microsystems Ltd, Germany) at 10× magnification. The number of adhering cells per unit area and total surface coverage of the materials by tissue cells were determined using Scion image analysis software, while the average area per spread cell was calculated from the ratio of the total surface coverage divided by the number of cells. All experiments were performed in six-fold on each implant material, comprising two sets of three independent experiments with modified culture medium, supplemented either with THB or TSB.

Tissue cell-bacteria co-culture experiments

Implant materials in Petri dishes were exposed to 10 μl suspensions (concentration 4 × 10 4 bacteria/ml) of the different bacterial strains in adhesion buffer for the streptococci and in PBS for the staphylococci and incubated at 37 °C, under 100% humidity for 60 min. Subsequently, the bacterial suspensions were removed by dipping three times in sterile buffer, yielding the presence of approximately 3 × 10 4 bacteria/cm 2 on the implant surfaces. Subsequently, HGF cells suspended in modified culture medium, supplemented with 2% of the appropriate bacterial growth medium, were seeded on bacterial-coated implant surfaces to a density of 20,000 cells/cm 2 . Bacteria and HGF cells were maintained at 37 °C in a humidified 5% CO 2 for 48 h. After 48 h, HGF cells were fixed, stained with TRITC-phalloidin and DAPI and analyzed as described above. The outcome of the race for the surface was expressed as the reduction in surface coverage by HGF cells in the presence of adhering bacteria. All experiments were performed in triplicate on each implant material.

Statistical analyses

To analyze differences in the water contact angle and surface roughness of the implant surfaces, values were compared by means of independent sample t -tests at a significance level of 0.05. Biofilm formation by the different bacterial strains and HGF cells interaction with the different implant materials in mono-culture experiments were compared by means of ANOVA. ANOVA was employed with Turkey’s HSD post hoc test using SigmaPlot version 12.0 software (Sytat Software, Inc, USA). If the normality test (Shapiro–Wilk) failed, Kruskal–Wallis one way analysis of variance on ranks was applied. A value of p < 0.05 was considered to be statistically significant. Outcomes of the race for the surface between the different strains and HGF cells were compared by means of ANOVA, based on the difference between cell surface coverage in absence and presence of adhering bacteria.

Materials and methods

Implant materials

All implant materials used in this study were provided by Institut Straumann AG (Basel, Switzerland) and were received as discs (diameter 5 mm, thickness 1 mm). Three different implant materials were included in this study, comprising titanium (Ti, cold-worked, grade 4), titanium-zirconium alloy (TiZr, 15% (wt) Zr) and zirconium oxide (ZrO 2 , Y-TZP). The materials were modified according to different procedures, indicated as

  • P: mirror-polished with finally a 0.04 μm SiO 2 suspension and cleaned by Deconex solution, followed by concentrated nitric acid and water.

  • M: ground to mimic the machined part of the implant and washed with Deconex solution, followed by concentrated nitric acid and water.

  • MA: ground and then acid-etched with a boiling mixture of concentrated HCl and H 2 SO 4 (or hot hydrofluoric acid in case of ZrO 2 ) and rinsed with concentrated nitric acid and water.

  • modMA: ground and acid-etched with HCl/H 2 SO 4 , as described above, but rinsed with water only under N 2 protection and directly stored individually in glass containers, filled with an isotonic NaCl solution, protected by N 2 filling.

M, MA, modMA and P modifications were all applied to Ti, whereas TiZr alloy and ZrO 2 were only modified according to M and modMA (TiZr) or MA (ZrO 2 ). M, MA and P modified discs were individually packed in aluminum foil and sealed in plastic.

All discs were sterilized in their respective packagings by γ-irradiation at 25–42 kGy.

Implant surface characterization

The hydrophobicity of the implant surfaces was determined by water contact angle measurements at room temperature with a home-made contour monitor, using the sessile drop (1–1.5 μl droplets) method. All values reported are averages over three different implant surfaces, while five droplets were placed on each disk.

The elemental surface composition of the implant surfaces was measured using an S-probe X-ray photoelectron spectrometer (Surface Science Instruments, Mountain View, CA, USA), equipped with an aluminum anode (10 kV, 22 mA) and a quartz monochromator. The direction of the photoelectron collection angle was 55 degrees with the sample surface and the electron flood gun was set at 10 eV. Survey scans were made with a 1000 μm × 250 μm spot and pass energy of 150 eV and used to calculate elemental surface compositions of the implant surfaces. The O 1s photoelectron binding energy peak was also recorded with a pass energy of 50 eV and decomposed into a component at 530.1 eV due to oxygen involved in oxide bonds and other components due to, for instance oxygen involved in carbonaceous contamination. All experiments were done on two discs of each implant material, while two separate measurements were taken on each sample.

The average surface roughness of the different implant surfaces was assessed with an atomic force microscope (Nanoscope IIIa Dimension™3100, Bruker, Santa Barbara, CA, USA), operated in the contact mode and using a Si 3 N 4 cantilever tip (DNP from Veeco, Woodbury, NY, USA) with a spring constant of 0.06 N/m. Analyses were done on three randomly selected places per sample disk, employing three discs for each implant material. The surface morphology of different implant surfaces was investigated using scanning electron microscopy (SEM). The specimens were fixed on SEM-stub-holders and visualized in a field emission scanning electron microscope (FE-SEM type 6301F, JEOL Ltd., Tokyo, Japan) at 2 kV and a magnification of 3000×.

Bacterial strains and culturing

For biofilm formation, S. oralis J22, S. mitis BMS, S. salivarius HB, and S. aureus ATCC 25923 were used. Strains were streaked onto blood agar plates and incubated for 24 h at 37 °C. Then, a fresh colony was inoculated in 10 ml of appropriate growth medium. S. oralis J22, S. mitis BMS, S. salivarius HB were inoculated in Todd Hewitt Broth (THB; OXOID, Basingstoke, England) and S. aureus ATCC 25923 was inoculated in Tryptone Soya Broth (TSB; OXOID, Basingstoke, England) for 24 h at 37 °C. Subsequently, 10 ml of the bacterial culture was inoculated into 200 ml growth medium and a second culture was grown for 16 h at 37 °C. Bacteria were harvested by centrifugation at 4000× g for 5 min at 10 °C, and streptococci washed twice with sterile adhesion buffer (2 mM potassium phosphate, 50 mM potassium chloride, and 1 mM calcium chloride, pH 6.8) while S. aureus ATCC 25923 was washed in sterile phosphate buffered saline (PBS, 10 mM potassium phosphate, 0.15 M NaCl, pH 7.0). Subsequently, bacteria were sonicated on ice (3 × 10 s) in sterile buffer at 30 W (Vibra Cell model 375; Sonics and Materials, Danbury, CT, USA) in order to break bacterial aggregates. Bacteria were counted in a Bürker–Türk counting chamber and the bacterial suspension was further diluted to a concentration of 3 × 10 5 bacteria per ml for all experiments.

Bacterial adhesion and biofilm formation

Bacterial adhesion and biofilm formation were studied on the bottom plate of a parallel plate flow chamber (175 mm × 17 mm × 0.75 mm). The bottom plate consisted of transparent poly (methyl methacrylate) in which six 1 mm deep inserts were prepared in the center region of the plate that could each hold one implant material disk (see Fig. 1 ). In this way, six different implant materials could be analyzed in one experiment, therewith reducing influence of biological variations between bacterial cultures. Poly (methyl methacrylate) bottom plates were cleaned in a 2% RBS detergent solution (Omniclean, Breda, The Netherlands) under sonication and thoroughly rinsed in demineralized water, 70% ethanol, water again and finally washed with sterile ultrapure water prior to use. The flow chamber used was equipped with heating elements and kept at 37 °C throughout an experiment. Bacterial deposition was observed with a CCD camera (Basler AG, Germany) mounted on a phase-contrast microscope Leica DM2000 (Leica Microsystems Ltd., Germany) with a 40× objective lens.

Nov 25, 2017 | Posted by in Dental Materials | Comments Off on Soft tissue integration versusearly biofilm formation on different dental implant materials
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