Abstract
Surface modification of biomaterials has been shown to improve the biological response to dental implants. The ability to create a controlled micro-texture on the implant via additive surface modification techniques with bioactive nanohydroxyapatite (nanoHA) may positively influence guided tissue regeneration.
Objective
The main goal of this study was to produce micro-fabricated SiO 2 surfaces modified with nanohydroxyapatite particles and to characterize their influence on the biological response of Human Dental-Pulp Mesenchymal Stem Cells (hDP-MSCs) and Streptococcus mutans .
Materials and methods
A combined methodology of sol–gel and soft-lithography was used to produce micropatterned SiO 2 thin films with different percentages of nanoHA micro-aggregates. The surfaces were characterized by SEM/EDS, FT-IR/ATR, AFM, XPS quantitative elemental percentage and contact angle measurements. Biological characterization was performed using hDP-MSCs cultures, while Streptococcus mutans was the selected microorganism to evaluate the bacterial adhesion on the thin films.
Results
Micropatterned SiO 2 surfaces with 0%, 1% and 5% of nanoHA micro-aggregates were successfully produced using a combination of sol–gel and soft-lithography. These surfaces controlled the biological response, triggering alignment and oriented proliferation of hDP-MSCs and significant differences in the adhesion of S. mutans to the different surfaces.
Significance
The micropatterned surfaces exhibited biocompatible behavior that induced an oriented adhesion and proliferation of hDP-MSCs while SiO 2 presented low bacterial adhesion. These results show that the combination of sol–gel with soft-lithography is a good approach to create micropatterned surfaces with bioactive nanoparticles for guided tissue regeneration.
1
Introduction
Titanium implants are the most common device used for teeth replacement based on their very high biocompatibility, excellent mechanical properties, osseointegration and long-term success . However, many studies have been reporting problems such as poor esthetics, low percentage of new bone formation, peri-implantitis and allergic reactions . These negative effects might require new phases of active therapy, increasing costs and decreasing the trust relationship between patient and dentist .
Ideally, any approach that results in increased tissue attachment and a reduced biofilm formation might reduce the risk of short and long-term failure in dental implantology. However, the development of implant surfaces that combine both effects is an enormous technological challenge if the complexity of hard and soft tissue/material interactions is considered.
In general, after an adequate primary stabilization, a chain of events must occur to achieve proper healing of soft and hard tissues around the implant. Some of these events such as cell adhesion, proliferation, organization, and differentiation might be modulated via micro-scale topography, but this modulation is cell-type dependent and does not take place for all cell types . The effects of this modulation are not well known for human dental-pulp mesenchymal stem cells (hDP-MSCs).
Another important factor for the success of implantology is the control of bacterial attachment and proliferation to prevent peri-implantitis development. Streptococcus mutans has been used as a model to study the bacterial adhesion patterns to dental materials and is the predominant colonizing microorganism of oral surfaces. Although little is known about its cooperation with others microorganisms and its biological effects on dental implants, a correlation between S. mutans and late colonizers responsible for peri-implantitis has been shown previously. Wang et al. demonstrated the interference on quorum sensing of S. mutans by periodontal pathogens such as Porphyromonas gingivalis and Treponema denticola , while Kumar et al. reported the existence of S. mutans in high abundance in a peri-implant crevice .
Recently, all-ceramic implants have been studied as an alternative treatment to improve esthetics in cases of patients with thin gingival biotype. Alumina, Zirconia, and their composites ( e.g. alumina toughened zirconia and zirconia toughened alumina) are commonly studied for orthopedic and dental applications ( i.e. : abutments, crowns, bridges, and implants) considering their well-documented biocompatibility, high fracture strength, light transmittance behavior, and color, which are desirable features to mimic the lost oral tissues .
Zirconia has been classified as a bioinert biomaterial that exhibits osseointegration comparable to that of titanium, as it has been reported in animal and clinical studies . However, long term clinical studies on this type of dental implants are not available. These ceramics are not free of long-term failures, as compared to titanium implants. After three decades of development of new surfaces for titanium implants, the mature technologies available exhibit many limitations for their implementation on zirconia implants. As a consequence, new technologies to modify all-ceramics implants are an open field for intensive research in dental materials.
The combination of soft-lithography and sol–gel chemistry was recently introduced to modify glass and 3Y-TZP surfaces . This synergy makes it possible to produce micropatterned surfaces with controlled chemistry, roughness, thickness and textures . These surface features have the potential to modulate early adhesion, alignment, proliferation and metabolic activity of osteoblast-like and human bone marrow stem cells (hBMSCs) .
Sol–gel has been increasingly employed for the processing of bioactive glasses since it exhibits characteristics such as low processing temperatures, controlled chemical composition and high homogeneity, allowing for the production of high purity SiO 2 films . On the other hand, soft-lithography is an inexpensive technique, amenable to a wide range of materials and processing environments, which can be used to create topographical patterns on a surface in a controlled and high throughput manner .
Several studies using patterned surfaces have shown improved cellular activity and enhancement of extracellular matrix synthesis of adherent cells, providing a faster and more reliable osseointegration response . Brunette’s group reported the use of micromachined titanium surfaces in vivo . The microtextured surfaces induced bone-like tissue formation after 6 weeks of subcutaneous implantation in the parietal area of rats, and partially mineralized globules after 4 weeks on calvarial explants, demonstrating that surface topography of an implant can promote better bone formation in vivo and in vitro .
Another relevant approach to improving the osseointegration and bone healing is the use of calcium phosphate materials as scaffolds, coatings, or bioactive particles integrated in coatings . Hydroxyapatite (HA) has been widely used as a biocompatible ceramic, mainly for contact with bone tissue. Preliminary studies with nanoHA micro-aggregates showed the ability to control protein adsorption and subsequent modulation/enhancement of osteoblast adhesion and long-term functionality .
The main goals of the current study were: to develop a fabrication technique for anisotropic micropatterned SiO 2 coatings containing bioactive particles; to carry out extensive materials characterization on such micropatterned surfaces; and to evaluate the in vitro cell/surface interactions with human dental-pulp MSCs and with bacteria. The tested hypotheses were that the addition of bioactive particles to anisotropic microstructured SiO 2 coatings would result in significant differences in terms of: (1) the in vitro adhesion and metabolic activity of hDP-MSCs; and (2) the adhesion of S. mutans .
2
Materials and methods
2.1
Thin films
Hybrid SiO 2 sols were produced via a sol–gel process with acid catalysis in a single stage, using Tetraethylorthosilicate (TEOS, Sigma–Aldrich, USA) and Methyltriethoxisilane (MTES, Sigma–Aldrich) as SiO 2 precursors . For all experiments, cover slips were used as a model substrate. Flat SiO 2 coatings were fabricated via spin-coating (SCS G3P-8 Specialty Coating Systems, Cookson Electronics, USA) at 3000 rpm for 45 s.
To produce the micropatterned SiO 2 coatings, a modified imprinting process was implemented based on soft lithography technologies ( Fig. 1 ). Initially, standard photolithography was used to produce a master model with desirable microfeatures (5 μm × 10 μm lines) in a clean room (Class 100) facility. Subsequently, negative polydimethylsiloxane (PDMS, Silastic T-2, Dow Corning, USA) replicas of the master were used to imprint SiO 2 suspensions with different concentrations of nanohydroxyapatite (nanoHA, Fluidinova SA, Portugal) micro-aggregates (0%, 1%, 5%). Finally, the samples were heat-treated at 500 °C for 60 min using a 5 °C/min ramp rate.
2.2
Materials characterization
Commercial nanoHA micro-aggregates were characterized in terms of particle size distribution and specific surface area using a Laser diffraction particle size analyzer (Master Sizer S; Malvern Instruments, UK) and the Brunauer–Emmett–Teller (BET) method (Monosorb Surface Area Analyzer MS-13, Quantachrome Corporation, UK).
Morphology, topography and bulk chemical analysis were carried out using SEM/EDS (FEI Quanta 400FEG ESEM/EDAX Genesis X4M). All samples were sputter-coated with palladium–gold.
Surface topography of the thin films was evaluated using Atomic Force Microscopy (Veeco Metrology Multimode/Nanoscope IVA, USA) in tapping mode. AFM images were acquired after the sintering treatment. Roughness was analyzed in terms of amplitude parameters (Rz and Rq) using a commercial software (NanoScope, Digital Instruments/Veeco, USA). The Rz parameter (height of the pattern) was calculated by analyzing three boxes of 7 μm × 45 μm in each image. In addition, the Root Mean Square (Rq) was calculated by analyzing nine boxes of 3 μm × 3 μm (on the top surface of the patterns.
The effects of the thermal cycle were evaluated on sintered and non-sintered SiO 2 thin films using a Fourier Transform Infrared Attenuated Total reflectance (FT-IR/ATR), with a Perkin-Elmer 2000 FT-IR spectrometer. Each sample was analyzed using a wavelength range of 4000–400 cm −1 at 4 cm −1 with 100 scans. Raw spectra were post-processed using a routine that included a smoothing filtering, baseline correction, and normalization (Spectrum 5.3.1, Perkin Elmer, USA).
Elemental surface analysis was conducted by X-ray photoelectron spectroscopy (XPS) using a digital system for data acquisition and analysis (ESCALAB 200A, VG Scientific with PISCES software, UK). All thin films were evaluated under the following conditions: data acquisition was performed with a pressure lower than 1 μPa and the X-rays were generated by an achromatic Aluminum Kα source operating at 15 kV (300 W). The spectrometer was calibrated with reference to Ag 3d 5/2 (368.27 eV) and operated in CAE mode with 20 eV pass energy.
All surfaces were analyzed within a depth of 5 nm with the photoelectrons being measured at a take off angle of 0°. Survey spectra were collected over a range of 0–1100 eV with analyzer pass energy of 50 eV. Spectra were post-processed using peak fitting with Gaussian–Lorentzian peak shape and linear type background subtraction using an XPS peak fitting program (XPS PEAK 4.1, R W. M. Kwok, China).
A contact angle device (OCA 15, DataPhysics Instruments GmbH, Germany) was used to quantify the surface hydrophobicity. The sessile drop method was applied with ultrapure water at 25 °C and the contact angle was calculated by the Laplace–Young function (SCA 20 software, DataPhysics Instruments GmbH). All the patterned surfaces were similarly oriented.
2.3
Biological characterization
2.3.1
Human dental-pulp MSCs cultures
Human dental pulp was obtained from an upper third molar extracted for orthodontic reasons. Informed consent was previously obtained to use this biological tissue that would be otherwise discarded. After an adequate disinfection procedure, the tooth was sectioned using a diamond cutter disc, chamber and channel pulp were removed and placed in a phosphate buffered saline (PBS; Gibco, UK) solution containing penicillin–streptomycin (1000 IU/ml and 25 μg/ml, Gibco, UK) and amphotericin B (25 μg/ml, Gibco, UK) for 15 min. The pulp was washed with sterile PBS and cut. Afterwards, the pulp chips were enzymatically digested using trypsin and collagenase. The primary culture was done in a tissue culture polystyrene (TCPS) Petri dish with α-minimal essential medium (α-MEM) containing 10% of fetal bovine serum, 1% of penicillin–streptomycin (100 IU/ml and 2.5 μg/ml, Gibco, UK), amphotericin B (2.5 μg/ml, Gibco, UK), and ascorbic acid (50 μg/ml, Sigma, USA). The cultures were incubated in a humidified atmosphere of 5% CO 2 at 37 °C and the medium was changed twice a week. When high confluence was reached (14–21 days), the adherent cells were washed with PBS and enzymatically released with 0.04% trypsin at 37 °C for 4 min and counted using a hemocytometer. The resultant cells were seeded at a density of 2 × 10 4 cells/cm 2 on the micropatterned samples and TCPS was used as a control. All cultures were incubated for 3 different time points (1, 7 and 14 days).
At each time point, the resazurin assay was used to determine the viability and proliferation of the cells. This is a simple and non-reactive assay, where a non-fluorescent blue component is reduced by the living cells to a pink fluorescent component. Fresh medium with 10% of resazurin was added to the cells and incubated for 3 h. Afterwards, 100 μl were transferred to a 96-well plate and the fluorescence was quantified in a microplate reader (Synergy HT, BioTek, USA) at 535 nm excitation wavelength and 590 nm emission wavelength. The results were expressed in relative fluorescence units (RFU). Subsequently, the cells were washed twice with pre-warmed PBS and fixed in a 10% (v/v) neutral buffered Formalin solution (Sigma, USA) for 15 min and morphologically evaluated by fluorescence microscopy and scanning electron microscopy (SEM).
For morphology evaluation with fluorescence microscopy (Zeiss Axiovert 200 M, Germany), the cells were washed and permeabilized with 0.1% (v/v) Triton X-100 (Sigma, USA) for 30 min. F-actin filaments were stained using Alexafluor phalloidin (Invitrogen, USA) for 30 min and the nuclei were stained with a buffer of Propidium iodide and RNase (BD Pharmigen, USA) for 10 min and washed with PBS. For the morphology evaluation via SEM, the cells were dehydrated in graded ethanol solutions and hexamethyldisilazane (HMDS, Ted Pella, USA) solutions from 50% to 100%, respectively . The samples were then sputter-coated with palladium–gold.
2.3.2
Bacterial cultures
A commercial strain of S. mutans DSM20523 (DSMZ, Germany) was cultivated in sterile tryptic soy broth (TSB, Difco, USA) for 48 h at 37 °C. The bacterial solution was harvested by centrifugation at 18 °C for 5 min at 2000 rpm and then washed twice and re-suspended in PBS solution at a concentration of 1.5 × 10 8 colony forming unit cells/ml, according to the McFarland standard, using a densitometer (BioMerieux, France). Flat SiO 2 , micropatterned samples and glass controls were incubated with 1 ml of the bacterial suspension, which has been previously prepared at 37 °C with gentle shaking for 90 min. Three replicates were used for each experiment. After incubation, each sample was gently rinsed twice with PBS to remove non-adherent or loosely adherent bacteria. Then, the samples were transferred into a tube with 5 ml of sterile PBS and sonicated for 1 s at 20 kHz (MS 73 probe, Sonopuls HD 2200, Bandelin, Germany). Finally, serial dilutions of the sonicated solutions were placed onto brain heart infusion (BHI, Liofilchem, Italy) culture plates and, after 48 h at 37 °C under microaerofilic conditions, the number of adherent bacteria was counted.
For the morphological analysis, after the washing step to remove non-adherent or barely adhered bacteria, the samples were fixed and prepared for SEM visualization as described above.
2.4
Statistical analysis
Triplicate experiments were performed. The results were expressed as the arithmetic mean ± standard deviation. The statistical analysis of the results was done using the one-way analysis of variance (One-way ANOVA) followed by the Tukey HSD post hoc test. Levels of p ≤ 0.05 were considered to be statistically significant. The statistical analysis was performed using the SPSS statistical software (Statistical Package for the Social Sciences Inc., USA).
2
Materials and methods
2.1
Thin films
Hybrid SiO 2 sols were produced via a sol–gel process with acid catalysis in a single stage, using Tetraethylorthosilicate (TEOS, Sigma–Aldrich, USA) and Methyltriethoxisilane (MTES, Sigma–Aldrich) as SiO 2 precursors . For all experiments, cover slips were used as a model substrate. Flat SiO 2 coatings were fabricated via spin-coating (SCS G3P-8 Specialty Coating Systems, Cookson Electronics, USA) at 3000 rpm for 45 s.
To produce the micropatterned SiO 2 coatings, a modified imprinting process was implemented based on soft lithography technologies ( Fig. 1 ). Initially, standard photolithography was used to produce a master model with desirable microfeatures (5 μm × 10 μm lines) in a clean room (Class 100) facility. Subsequently, negative polydimethylsiloxane (PDMS, Silastic T-2, Dow Corning, USA) replicas of the master were used to imprint SiO 2 suspensions with different concentrations of nanohydroxyapatite (nanoHA, Fluidinova SA, Portugal) micro-aggregates (0%, 1%, 5%). Finally, the samples were heat-treated at 500 °C for 60 min using a 5 °C/min ramp rate.
2.2
Materials characterization
Commercial nanoHA micro-aggregates were characterized in terms of particle size distribution and specific surface area using a Laser diffraction particle size analyzer (Master Sizer S; Malvern Instruments, UK) and the Brunauer–Emmett–Teller (BET) method (Monosorb Surface Area Analyzer MS-13, Quantachrome Corporation, UK).
Morphology, topography and bulk chemical analysis were carried out using SEM/EDS (FEI Quanta 400FEG ESEM/EDAX Genesis X4M). All samples were sputter-coated with palladium–gold.
Surface topography of the thin films was evaluated using Atomic Force Microscopy (Veeco Metrology Multimode/Nanoscope IVA, USA) in tapping mode. AFM images were acquired after the sintering treatment. Roughness was analyzed in terms of amplitude parameters (Rz and Rq) using a commercial software (NanoScope, Digital Instruments/Veeco, USA). The Rz parameter (height of the pattern) was calculated by analyzing three boxes of 7 μm × 45 μm in each image. In addition, the Root Mean Square (Rq) was calculated by analyzing nine boxes of 3 μm × 3 μm (on the top surface of the patterns.
The effects of the thermal cycle were evaluated on sintered and non-sintered SiO 2 thin films using a Fourier Transform Infrared Attenuated Total reflectance (FT-IR/ATR), with a Perkin-Elmer 2000 FT-IR spectrometer. Each sample was analyzed using a wavelength range of 4000–400 cm −1 at 4 cm −1 with 100 scans. Raw spectra were post-processed using a routine that included a smoothing filtering, baseline correction, and normalization (Spectrum 5.3.1, Perkin Elmer, USA).
Elemental surface analysis was conducted by X-ray photoelectron spectroscopy (XPS) using a digital system for data acquisition and analysis (ESCALAB 200A, VG Scientific with PISCES software, UK). All thin films were evaluated under the following conditions: data acquisition was performed with a pressure lower than 1 μPa and the X-rays were generated by an achromatic Aluminum Kα source operating at 15 kV (300 W). The spectrometer was calibrated with reference to Ag 3d 5/2 (368.27 eV) and operated in CAE mode with 20 eV pass energy.
All surfaces were analyzed within a depth of 5 nm with the photoelectrons being measured at a take off angle of 0°. Survey spectra were collected over a range of 0–1100 eV with analyzer pass energy of 50 eV. Spectra were post-processed using peak fitting with Gaussian–Lorentzian peak shape and linear type background subtraction using an XPS peak fitting program (XPS PEAK 4.1, R W. M. Kwok, China).
A contact angle device (OCA 15, DataPhysics Instruments GmbH, Germany) was used to quantify the surface hydrophobicity. The sessile drop method was applied with ultrapure water at 25 °C and the contact angle was calculated by the Laplace–Young function (SCA 20 software, DataPhysics Instruments GmbH). All the patterned surfaces were similarly oriented.
2.3
Biological characterization
2.3.1
Human dental-pulp MSCs cultures
Human dental pulp was obtained from an upper third molar extracted for orthodontic reasons. Informed consent was previously obtained to use this biological tissue that would be otherwise discarded. After an adequate disinfection procedure, the tooth was sectioned using a diamond cutter disc, chamber and channel pulp were removed and placed in a phosphate buffered saline (PBS; Gibco, UK) solution containing penicillin–streptomycin (1000 IU/ml and 25 μg/ml, Gibco, UK) and amphotericin B (25 μg/ml, Gibco, UK) for 15 min. The pulp was washed with sterile PBS and cut. Afterwards, the pulp chips were enzymatically digested using trypsin and collagenase. The primary culture was done in a tissue culture polystyrene (TCPS) Petri dish with α-minimal essential medium (α-MEM) containing 10% of fetal bovine serum, 1% of penicillin–streptomycin (100 IU/ml and 2.5 μg/ml, Gibco, UK), amphotericin B (2.5 μg/ml, Gibco, UK), and ascorbic acid (50 μg/ml, Sigma, USA). The cultures were incubated in a humidified atmosphere of 5% CO 2 at 37 °C and the medium was changed twice a week. When high confluence was reached (14–21 days), the adherent cells were washed with PBS and enzymatically released with 0.04% trypsin at 37 °C for 4 min and counted using a hemocytometer. The resultant cells were seeded at a density of 2 × 10 4 cells/cm 2 on the micropatterned samples and TCPS was used as a control. All cultures were incubated for 3 different time points (1, 7 and 14 days).
At each time point, the resazurin assay was used to determine the viability and proliferation of the cells. This is a simple and non-reactive assay, where a non-fluorescent blue component is reduced by the living cells to a pink fluorescent component. Fresh medium with 10% of resazurin was added to the cells and incubated for 3 h. Afterwards, 100 μl were transferred to a 96-well plate and the fluorescence was quantified in a microplate reader (Synergy HT, BioTek, USA) at 535 nm excitation wavelength and 590 nm emission wavelength. The results were expressed in relative fluorescence units (RFU). Subsequently, the cells were washed twice with pre-warmed PBS and fixed in a 10% (v/v) neutral buffered Formalin solution (Sigma, USA) for 15 min and morphologically evaluated by fluorescence microscopy and scanning electron microscopy (SEM).
For morphology evaluation with fluorescence microscopy (Zeiss Axiovert 200 M, Germany), the cells were washed and permeabilized with 0.1% (v/v) Triton X-100 (Sigma, USA) for 30 min. F-actin filaments were stained using Alexafluor phalloidin (Invitrogen, USA) for 30 min and the nuclei were stained with a buffer of Propidium iodide and RNase (BD Pharmigen, USA) for 10 min and washed with PBS. For the morphology evaluation via SEM, the cells were dehydrated in graded ethanol solutions and hexamethyldisilazane (HMDS, Ted Pella, USA) solutions from 50% to 100%, respectively . The samples were then sputter-coated with palladium–gold.
2.3.2
Bacterial cultures
A commercial strain of S. mutans DSM20523 (DSMZ, Germany) was cultivated in sterile tryptic soy broth (TSB, Difco, USA) for 48 h at 37 °C. The bacterial solution was harvested by centrifugation at 18 °C for 5 min at 2000 rpm and then washed twice and re-suspended in PBS solution at a concentration of 1.5 × 10 8 colony forming unit cells/ml, according to the McFarland standard, using a densitometer (BioMerieux, France). Flat SiO 2 , micropatterned samples and glass controls were incubated with 1 ml of the bacterial suspension, which has been previously prepared at 37 °C with gentle shaking for 90 min. Three replicates were used for each experiment. After incubation, each sample was gently rinsed twice with PBS to remove non-adherent or loosely adherent bacteria. Then, the samples were transferred into a tube with 5 ml of sterile PBS and sonicated for 1 s at 20 kHz (MS 73 probe, Sonopuls HD 2200, Bandelin, Germany). Finally, serial dilutions of the sonicated solutions were placed onto brain heart infusion (BHI, Liofilchem, Italy) culture plates and, after 48 h at 37 °C under microaerofilic conditions, the number of adherent bacteria was counted.
For the morphological analysis, after the washing step to remove non-adherent or barely adhered bacteria, the samples were fixed and prepared for SEM visualization as described above.
2.4
Statistical analysis
Triplicate experiments were performed. The results were expressed as the arithmetic mean ± standard deviation. The statistical analysis of the results was done using the one-way analysis of variance (One-way ANOVA) followed by the Tukey HSD post hoc test. Levels of p ≤ 0.05 were considered to be statistically significant. The statistical analysis was performed using the SPSS statistical software (Statistical Package for the Social Sciences Inc., USA).
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