First titanium dental implants with white surfaces: Preparation and in vitrotests

Abstract

Objectives

To evaluate first white titanium surfaces developed for improvement of existing clinically used titanium-based implants Ticer.

Methods

The anodic plasma-electrochemical oxidation in aqueous solutions of sodium hydroxide and calcium dihydrogen phospate was used to prepare three novel anodic conversion layers with white titanium oxide surfaces. The surfaces have been characterized by the means of scanning electron microscopy, surface microanalysis and X-ray diffraction. In vitro studies were conducted on primary human osteoblast cells using novel surfaces (M1–M3) as well as commercially pure titanium (Ti cp), Ticer and SS (subtracted surface). An indirect toxicity test using MTT and SRB assays has been carried out. Furthermore, immunohistochemical analysis of cell proliferation, morphology, and expression of non-collagenous bone matrix proteins (sialoprotein, BSP, and osteocalcin, OC) were performed.

Results

The basic morphology of the surfaces shows clusters in a size of 100 μm of knob-like structures. The coatings are composed of rutile and monoclinic sodium titanates. Novel white surfaces (M1–M3) induced proliferation rates, morphological changes and influenced the expression of OC and BSP similarly to Ticer. On the other hand, Ti cp and SS exhibited different in vitro behavior.

Significance

The novel surfaces expressed similar in vitro behavior as Ticer, successfully used in clinical practice. Furthermore, due to their white color they are also promising from the esthetic point of view. The results described herein open the door toward a new generation of white titanium dental implants.

Introduction

The main purpose for development of dental implants is to stimulate osseointegration of the implant with the surrounding bone and maintenance and functional restoration of the existing bone . Surface characteristics of biomaterials, such as surface topography, surface charges, components, chemical states and wettability have influence on the interactions of bone matrix and osteoblasts with biomaterials . Dental implants with modified surfaces have been obtained employing different methods, e.g. grit blasting, acid etching, anodization, plasma spraying or by hybrid techniques (sand blast with large grit and acid etching) . These modifications improve bone-to-implant contact and therefore long-term success of dental implants. The different implant surface modification methods may lead to diverse and distinctive surface properties that might influence the host-to-implant response.

After a surgical implantation procedure, implant surface is in direct contact with the blood and serum, from which it might absorb proteins and then cell colonization is occurring . Osteoblasts-implant interaction comprises of the formation of the initial cell contact, followed by adhesion and spreading of cells on the material surface .

The cellular response and the type of primary interactions considerably affect the proliferation and differentiation of the osteoblast cells .

As a biomedical metal implants, titanium-based ones have exceptional surface properties . Additionally, due to formation of a thin layer of titanium dioxide, they are highly resistant to corrosion and have good biocompatibility, durability and strength . Specific surface handling of titanium-based implants is required for a successful osseointegration process . Commonly, rough, textured and porous surfaces stimulate cell attachment, differentiation and the formation of extracellular matrix .

Unfortunately, the main disadvantage of titanium implants reflects in their dark grayish color, the fact that might cause esthetic issues for patients especially in front teeth area . The most part of the implant is embedded within the bone but the appearance of dark shimmer of titanium implants might occur, especially when the soft peri-implant mucosa is of thin biotype or recedes over time. In order to improve this esthetic aspect many efforts have been taken in order to develop tooth-colored implants. Nowadays, zirconia (zirconium dioxide) has been used as alternative material to titanium for the fabrication of dental implants. Those implants mimic natural teeth with their white color and also show remarkable biocompatibility. However, their smooth surfaces are not beneficial for osseointegration due to a poor interaction with tissues. Surface modification of zirconia implants is difficult, very often accompanied with surface damage and disruption, which might lead to fracture of implants .

Understanding of the influence of biomaterial surface topography on cellular behavior is important to improve healing of implants . Herein, three titanium-based implants obtained by anodic plasma-electrochemical oxidation with well-defined structure were used for the evaluation of cellular behavior of osteoblasts in response to surface topographical effects. The modification of titanium-based materials was characterized by the means of scanning electron microscopy, surface microanalysis and X-ray diffraction. The effect of surface topography on osteoblasts proliferation, spreading and expression of bone sialoprotein and osteocalcin were evaluated.

Materials and methods

Coating process and surface characterization

The reference material Ticer and Ti cp were obtained as kind gift from ZL Microdent, Germany. Ti cp was employed as base material for the anodic plasma-electrochemical coating process. All anodic conversion coatings were prepared in an electrochemical cell using an electrical pulse generator as power supply . The titanium samples acted as the anode while a platinum wire constituted the cathode. The commercial used Ticer-coating is generated in Ca(H 2 PO 4 ) 2 electrolyte . As electrolytes for the preparation of M1–M3 aqueous solutions of 0.02 M Ca(H 2 PO 4 ) 2 (pH 3) and 1.25 M NaOH were used. M1 was obtained by coating of Ti cp in 1.25 M NaOH electrolyte. Sample M2 was coated twice, first in the 1.25 M NaOH electrolyte and second in the 0.02 M Ca(H 2 PO 4 ) 2 electrolyte. Sample M3 corresponds sample M1 with an additional soaking in a 0.02 M Ca(H 2 PO 4 ) 2 solution for the infiltration of calcium and phosphate ions.

The electrolytes were continuously magnetically stirred in a 250 ml double-wall glass beaker. The electrolyte temperature of 30 °C was thermostatically controlled. Before the coating process all titanium samples were chemically polished for 10 s in a mixture of phosphorus, hydrofluoric and nitric acid (H 3 PO 4 /HF/HNO 3 ) in a ratio of 49/24/27% (v/v/v), and ultrasonically cleaned in water followed by 2-propanol rinsing. The current density during the anodic plasma-electrochemical oxidation was up to 0.6 A/cm 2 . For the preparation of the SS (subtracted surface) a mixture of equal parts of concentrated hydrochloric acid and sulphuric acid was used for etching. After the coating processes, the specimens were rinsed in distilled water and 2-propanol, dried and stored in air.

For characterization, the surface topography of the specimens was examined by a JEOL JSM 840A scanning electron microscope (SEM). Surface microanalysis to determine the concentration of the chemical elements was carried out with energy dispersive spectroscopy (EDS). X-ray diffraction was used for the determination of solid state phases of the anodic conversion layers.

Cell culture

All procedures used in this study were approved by the Ethics Committee of the University of Leipzig (No. 086-2008) and performed according to the rules of the Declaration of Helsinki from 1975 (revised in 1983). Human mandibular bone samples without any clinical or radiographic pathological evidence were obtained from one male donor, who was undergoing lower wisdom tooth surgery at the Department of Oral, Maxillary, Facial and Reconstructive Plastic Surgery at the University Hospital of Leipzig. The bone sample was placed in a sterile tube containing 0.05 M sterile phosphate buffered saline (PBS) at pH 7.4, and penicillin/streptomycin at 100 IU/ml each (PromoCell, Heidelberg, Germany). Subsequently, all samples were processed under sterile conditions. The bone samples were cut into 0.1 × 0.1 cm pieces. After rinsing several times in PBS, the material was incubated with 0.25% collagenase type IV (166 U/mg; Biochrom, Berlin, Germany) for 30 min at 37 °C. Afterwards, suspension was discharged and the rest incubated for 2 h in the presence of 0.25% collagenase type IV at 37 °C. Then, the cells were washed, centrifuged (300 × g for 10 min) and cultured in Osteoblast growth medium (PromoCell) and supplemented with 10% fetal bovine serum (PromoCell) in an atmosphere of 5% CO 2 at 37 °C. The medium was changed twice a week, and cells were grown to confluence in culture flasks (Greiner Bio-One, Frickenhausen, Germany). Thereafter, the cells were subcultured from initially isolated primary cells and seeded at a density of 4000 cells/ml (2000 cells/well) in chamber slides or 96 well plates .

Experimental design

The 96 well plates were used for cytotoxicity and eight chamber slides for all other experiments. Six sterile implant materials (round discs: diameter 6 mm, thickness 0.5 mm) were tested. Thus, three novel materials (M1–M3) and three referent clinically-employed materials (Ti cp, Ticer and SS) were exanimated. Cell cultivated on glass were used as control group. Each experiment was performed in triplicate. For each immunocytochemistry assay one eight chamber slide with the osteoblast cells was removed from the incubator on days 3, 5, 7 or 10 and the cells were fixed in paraformaldehyde (4% in PBS) for 15 min and rinsed in PBS.

In vitro cytotoxicity

An indirect cytotoxicity test using MTT and SRB colorimetric assays has been carried out. The metabolic activity (MTT) of cells and total protein amount (SRB), previously incubated with material extracts (extraction ratio: 3 cm 2 /ml) for 72 h, were determined. Absorbance was measured at 570 nm using a 96 well plate reader (Tecan Spectra, Crailsheim, Germany). Results are presented as percentage absorption value ( S ) in comparison to the control group.

DAPI staining of the cells

Cells attached to the glass or materials from the eight chamber slide were rinsed several times with PBS. Then, the cell nuclei were stained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI; Serva, Heidelberg, Germany) in order to quantify cell proliferation as the number of cells on the investigated surfaces .

Morphology of the cells

The cells were stained with acridine orange (AO, 15 μl, 3 μg/ml) and the cell morphology was determined . In short, cell morphology was evaluated by measuring the footprint area of the cell on the surface after attachment and using a shape factor, ϕ = (4π A )/ p 2 ( A = footprint area; p = the perimeter of the cell).

BSP and OC expression

For the immunocytochemical estimation of bone sialo protein (BSP) and osteocalcin (OC), the attached cells were treated for 2 h with 10% normal goat serum (Vector, Burlingame, CA, USA) in PBS and incubated overnight at 4 °C with 1:100 diluted primary antibodies against BSP (monoclonal, mouse-anti-human; Immundiagnostik AG, Bensheim, Germany) or osteocalcin (monoclonal, mouse-anti-human; Acris, Hiddenhausen, Germany). After washing in PBS, the bound primary antibodies were visualized by incubation for 2 h with 1:50 diluted goat-antimouse-Cy3 (Jackson Immuno Research, West Grove, PA, USA) in PBS containing 4% bovine serum albumin (Serva). After rinsing several times in PBS, cell preparations were counterstained using DAPI (Serva) and coverslipped.

Observation methods

A motorized Zeiss Axiophot2 microscope (Zeiss, Oberkochen, Germany) equipped with the appropriate filters was used to determine proliferation, cell shape, as well as to observe BSP and OC expression. In order to quantify cell number, DAPI-labeled cells were counted on the edge and middle, six and four images respectively, of each surface topography and glass. Sections were screened at 200× magnification. The same imaging procedure was applied for determination of BSP and OC expression. For evaluation of cell morphology more than 60 single cells were randomly chosen. All images were analyzed using Image J software (Version 1.43u for Windows).

Statistics

Data from the repeated experiments are presented as the mean and standard deviation. The significance of the differences between various treatments was assessed by ANOVA followed by the Student–Newman–Keuls test. Differences were considered significant if the p value was lower than 0.05.

Materials and methods

Coating process and surface characterization

The reference material Ticer and Ti cp were obtained as kind gift from ZL Microdent, Germany. Ti cp was employed as base material for the anodic plasma-electrochemical coating process. All anodic conversion coatings were prepared in an electrochemical cell using an electrical pulse generator as power supply . The titanium samples acted as the anode while a platinum wire constituted the cathode. The commercial used Ticer-coating is generated in Ca(H 2 PO 4 ) 2 electrolyte . As electrolytes for the preparation of M1–M3 aqueous solutions of 0.02 M Ca(H 2 PO 4 ) 2 (pH 3) and 1.25 M NaOH were used. M1 was obtained by coating of Ti cp in 1.25 M NaOH electrolyte. Sample M2 was coated twice, first in the 1.25 M NaOH electrolyte and second in the 0.02 M Ca(H 2 PO 4 ) 2 electrolyte. Sample M3 corresponds sample M1 with an additional soaking in a 0.02 M Ca(H 2 PO 4 ) 2 solution for the infiltration of calcium and phosphate ions.

The electrolytes were continuously magnetically stirred in a 250 ml double-wall glass beaker. The electrolyte temperature of 30 °C was thermostatically controlled. Before the coating process all titanium samples were chemically polished for 10 s in a mixture of phosphorus, hydrofluoric and nitric acid (H 3 PO 4 /HF/HNO 3 ) in a ratio of 49/24/27% (v/v/v), and ultrasonically cleaned in water followed by 2-propanol rinsing. The current density during the anodic plasma-electrochemical oxidation was up to 0.6 A/cm 2 . For the preparation of the SS (subtracted surface) a mixture of equal parts of concentrated hydrochloric acid and sulphuric acid was used for etching. After the coating processes, the specimens were rinsed in distilled water and 2-propanol, dried and stored in air.

For characterization, the surface topography of the specimens was examined by a JEOL JSM 840A scanning electron microscope (SEM). Surface microanalysis to determine the concentration of the chemical elements was carried out with energy dispersive spectroscopy (EDS). X-ray diffraction was used for the determination of solid state phases of the anodic conversion layers.

Cell culture

All procedures used in this study were approved by the Ethics Committee of the University of Leipzig (No. 086-2008) and performed according to the rules of the Declaration of Helsinki from 1975 (revised in 1983). Human mandibular bone samples without any clinical or radiographic pathological evidence were obtained from one male donor, who was undergoing lower wisdom tooth surgery at the Department of Oral, Maxillary, Facial and Reconstructive Plastic Surgery at the University Hospital of Leipzig. The bone sample was placed in a sterile tube containing 0.05 M sterile phosphate buffered saline (PBS) at pH 7.4, and penicillin/streptomycin at 100 IU/ml each (PromoCell, Heidelberg, Germany). Subsequently, all samples were processed under sterile conditions. The bone samples were cut into 0.1 × 0.1 cm pieces. After rinsing several times in PBS, the material was incubated with 0.25% collagenase type IV (166 U/mg; Biochrom, Berlin, Germany) for 30 min at 37 °C. Afterwards, suspension was discharged and the rest incubated for 2 h in the presence of 0.25% collagenase type IV at 37 °C. Then, the cells were washed, centrifuged (300 × g for 10 min) and cultured in Osteoblast growth medium (PromoCell) and supplemented with 10% fetal bovine serum (PromoCell) in an atmosphere of 5% CO 2 at 37 °C. The medium was changed twice a week, and cells were grown to confluence in culture flasks (Greiner Bio-One, Frickenhausen, Germany). Thereafter, the cells were subcultured from initially isolated primary cells and seeded at a density of 4000 cells/ml (2000 cells/well) in chamber slides or 96 well plates .

Experimental design

The 96 well plates were used for cytotoxicity and eight chamber slides for all other experiments. Six sterile implant materials (round discs: diameter 6 mm, thickness 0.5 mm) were tested. Thus, three novel materials (M1–M3) and three referent clinically-employed materials (Ti cp, Ticer and SS) were exanimated. Cell cultivated on glass were used as control group. Each experiment was performed in triplicate. For each immunocytochemistry assay one eight chamber slide with the osteoblast cells was removed from the incubator on days 3, 5, 7 or 10 and the cells were fixed in paraformaldehyde (4% in PBS) for 15 min and rinsed in PBS.

In vitro cytotoxicity

An indirect cytotoxicity test using MTT and SRB colorimetric assays has been carried out. The metabolic activity (MTT) of cells and total protein amount (SRB), previously incubated with material extracts (extraction ratio: 3 cm 2 /ml) for 72 h, were determined. Absorbance was measured at 570 nm using a 96 well plate reader (Tecan Spectra, Crailsheim, Germany). Results are presented as percentage absorption value ( S ) in comparison to the control group.

DAPI staining of the cells

Cells attached to the glass or materials from the eight chamber slide were rinsed several times with PBS. Then, the cell nuclei were stained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI; Serva, Heidelberg, Germany) in order to quantify cell proliferation as the number of cells on the investigated surfaces .

Morphology of the cells

The cells were stained with acridine orange (AO, 15 μl, 3 μg/ml) and the cell morphology was determined . In short, cell morphology was evaluated by measuring the footprint area of the cell on the surface after attachment and using a shape factor, ϕ = (4π A )/ p 2 ( A = footprint area; p = the perimeter of the cell).

BSP and OC expression

For the immunocytochemical estimation of bone sialo protein (BSP) and osteocalcin (OC), the attached cells were treated for 2 h with 10% normal goat serum (Vector, Burlingame, CA, USA) in PBS and incubated overnight at 4 °C with 1:100 diluted primary antibodies against BSP (monoclonal, mouse-anti-human; Immundiagnostik AG, Bensheim, Germany) or osteocalcin (monoclonal, mouse-anti-human; Acris, Hiddenhausen, Germany). After washing in PBS, the bound primary antibodies were visualized by incubation for 2 h with 1:50 diluted goat-antimouse-Cy3 (Jackson Immuno Research, West Grove, PA, USA) in PBS containing 4% bovine serum albumin (Serva). After rinsing several times in PBS, cell preparations were counterstained using DAPI (Serva) and coverslipped.

Observation methods

A motorized Zeiss Axiophot2 microscope (Zeiss, Oberkochen, Germany) equipped with the appropriate filters was used to determine proliferation, cell shape, as well as to observe BSP and OC expression. In order to quantify cell number, DAPI-labeled cells were counted on the edge and middle, six and four images respectively, of each surface topography and glass. Sections were screened at 200× magnification. The same imaging procedure was applied for determination of BSP and OC expression. For evaluation of cell morphology more than 60 single cells were randomly chosen. All images were analyzed using Image J software (Version 1.43u for Windows).

Statistics

Data from the repeated experiments are presented as the mean and standard deviation. The significance of the differences between various treatments was assessed by ANOVA followed by the Student–Newman–Keuls test. Differences were considered significant if the p value was lower than 0.05.

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Nov 25, 2017 | Posted by in Dental Materials | Comments Off on First titanium dental implants with white surfaces: Preparation and in vitrotests
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