Abstract
Objectives
A tight seal between the epithelium and the dental implant surface is required to prevent bacterial inflammation and soft tissue recession and therefore to demonstrate a long-term success. Surface hydrophilicity was recently shown to promote osseointegration. The aim of this study was to investigate the influence of surface hydrophilicity in combination with surface topography of Ti implant surfaces on the behavior and activation/differentiation of epithelial cells using a set of in vitro experiments mimicking the implant-soft tissue contact.
Methods
Hydrophobic acid-etched (A) and coarse-grit-blasted, acid-etched (SLA) surfaces and hydrophilic acid-etched (modA) and modSLA surfaces were produced. The behavior of an oral squamous cell carcinoma cell line (HSC-2) grown on all surfaces was compared through determination of cell attachment and proliferation/viability (CCK-8 and MTT assay), time-lapse microscopy of fluorescence labeled cells and determination of gene expression by real time polymerase chain reaction.
Results
Within the surfaces with similar wettability cell spreading and cell movements observed by time-lapse microscopy after one day of incubation were most pronounced on smoother (A and modA) surfaces compared to rougher (SLA and modSLA) surfaces. Within the surfaces with similar roughness the hydrophilic surfaces (modA and modSLA) showed more cell spreading and cell activity compared to the hydrophobic surfaces (A and SLA).
The relative gene expressions of cytokeratin14, integrin α6, integrin β4, vinculin, transforming growth factor (TGF)-β, TGF-β1, and TGF-β3 were decreased in HSC-2 on all four types of Ti surfaces compared to control surfaces (tissue culture polystyrene; p < 0.01) and there was no significant difference of gene expression on the four different implant-surfaces.
Significance
We have demonstrated that for proliferation and spreading of HSC-2 cells the smoother and hydrophilic surface is optimal (modA). These results suggest that surface hydrophilicity might positively influence the epithelial seal around dental implants. All tested titanium surfaces downregulate cell attachment, cell proliferation, expression of adhesion promoters, and cytokines involved in wound healing in HSC-2 cells compared to control surfaces.
1
Introduction
Dental implants are widely used and titanium is recognized as an outstanding biomaterial which has a good biocompatibility to the body. For a long time, only osseointegration was identified as a local factor that may interfere with the success of dental implants . It is well known that not only osseointegration of dental implants contributes to wound healing, but also the soft tissue adjacent to the implant . Among these soft tissues, epithelium is crucial for providing a biological seal of the implant which also determines its success. More and more in vitro and in vivo studies began to focus on the interactions of epithelium or epithelial cells and different titanium implant surfaces .
In the past 3 decades, many efforts have been devoted to the investigation of surface topography effects, such as sand-blasting or acid-etching and combinations thereof . Osseointegration was more pronounced on rough surfaces . The connective tissue around Ti implants prefers smooth surfaces . Various studies have been performed investigating the proliferation of epithelial cells on smooth and rough titanium surfaces. In 1978, Baumhammers reported that the gingival epithelial cells grew equally well on either smooth or rough (sand-blasted) surfaces . Years later it was found that epithelial cells attached and spread more readily on smooth than on rough sandblasted titanium surfaces and smooth titanium biomaterial surfaces are optimal for epithelial cell adhesion and spreading . One study using epidermal keratinocytes reported that epithelial cells proliferated on controls and smooth titanium surfaces after a lag period, but not on rough titanium surfaces . In 2009, Werner et al. reported about a microstructured titanium material made of assembled smooth titanium beads. This smooth titanium surface was very well colonized by epithelial cells in vitro and by cells of gingival soft tissue in vivo . In the past few years hydrophilic/hydrophobic surfaces which change the implant surface chemistry that influence surface charge and wettability were produced . Modified-SLA (sand-blasted and acid-etched, hydrophilic) surfaces showed an advantage by improving osteoblast activity in vitro , and also in vivo significant greater bone-to-implant contacts at 2 and 4 weeks of healing compared to the standard surface . An in vivo study was performed to investigate the effects of surface hydrophilicity on soft and hard tissue integration at non-submerged titanium implants. The results show that epithelial cells appeared to be in close contact with hydrophilic surfaces after 14 days of healing. This study was performed in dogs and only described the early effects of surface hydrophilicity on epithelial cells without investigating any possible mechanisms for this effect. Nevertheless, there are no data about the behavior of epithelial cell growing on hydrophilic titanium surface in vitro and there is no systematic investigation of any possible mechanisms for the influence of surface hydrophilicity on epithelial cells.
Dental implants with a high success rate require a tight seal between the epithelium and the surface to prevent bacterial inflammation and soft tissue recession . For the purpose of sealing the internal environment, the formation of a peri-implant junctional epithelium (JE) which is nearly the same structure as the dental junctional epithelium is needed. To know more about the physiology and pathology of peri-implant tissues it is necessary to investigate the wound healing procedure of soft tissues, especially functional cytokines which are expressed by epithelial cells and seem to play an active role to the JE attachment. Cytokeratins (CKs) are basal cell markers in human stratified squamous epithelia and it was shown that CK14 was expressed throughout the JE . Integrins are a family of transmembrane extracellular matrix receptors . The integrin α6β4 is considered to function as a receptor for Laminin-5 and to form tight adhesive junctions between cells and the basement membrane by being a component of type I or type II hemidesmosomes . In culture, most cells adhere to their underlying growth surface by means of focal contacts. Focal contacts can be identified by the presence of the actin binding protein vinculin . Transforming growth factor-β (TGF-β), regulating cell growth and differentiation, is expressed in several cell types including epithelial cells. TGF-β has a broad spectrum of activities such as effects on epithelial cell proliferation and differentiation .
The aims of the present study were (i) to evaluate if the different topographies and chemistries of Ti surfaces can modify the proliferation and the expression of various cytokines of the epithelial cells involved in the wound healing process, including CK14, vinculin, integrin α6β4, TGF-β, TGF-β1, and TGF-β3 and (ii) to observe the behavior of epithelial cells growth on those surfaces during long time incubation by time-lapse microscopy.
In line with the aims of this study the following null-hypotheses were formulated: Surface hydrophilicity and surface topography of Ti implant surfaces do not promote proliferation or spreading of epithelial cells. Surface hydrophilicity and surface topography of Ti implant surfaces do not enhance the expression of various cytokines involved in wound healing.
2
Materials and methods
2.1
Preparation of titanium disks
Commercially pure Ti disks (grade 2, Institut Straumann AG, Basel, Switzerland) were characterized by cylindrical shape with a diameter of 15 mm and 1 mm in thickness.
Surface chemistry is a variable for peri-implant healing since it influences surface energy and wettability and therefore the behavior of epithelial cells . Increased wettability enhances interaction between the implant surface and the biologic environment . More recently, an advanced technology of improving surface energy and wettability has been created to modify surface chemistry of Ti implants . Surface roughness is an other variable influencing the behavior of epithelial cells .
In this study, four kinds of surfaces were used: hydrophobic acid-etched (A) surface, modified [hydrophilic] acid-etched (modA) surface, hydrophobic coarse-grit-blasted and acid-etched (SLA) surface and modified [hydrophilic] coarse-grit-blasted and acid-etched (modSLA) surface. The preparation of Ti disks has been described previously . The chemical composition of all surfaces was determined by X-ray photoelectron spectroscopy whereas surface topography and roughness were characterized using scanning electron microscopy and white light confocal microscope, respectively. Hydrophilicity and contact angle hysteresis were tensiometriclly examined by the Wilhelmy method by means of an electrobalance .
2.2
Cell culture
HSC-2 (Oral squamous cell carcinoma cell line, Japan Health Sciences Foundation, Health Science Research Resources Bank, JCRBD 6222) cells were used as a model to investigate the effects of surface and material variations on epithelial cells. The HSC-2 cultures were maintained in MEM medium (Gibco, Austria), supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 10% FCS. Cultures were subdivided by using Trypsin–EDTA solution (1:250, PAA, Austria). All experiments were performed using cells between fifth and eighth passage. The culture medium was changed every 2 days.
2.3
Cell proliferation
The Ti disks were placed in 24-well tissue culture plates (TPP, Switzerland) and then 1 × 10 5 cells were added to each well and were incubated at 37 °C and 5% CO 2 in a humidified atmosphere. In every experiment, six wells with each of the four types of Ti disks and six wells with no disks (tissue culture polystyrene [TCPS] controls) were used. All subsequent experiments were repeated three times.
2.4
CCK-8 (Cell Counting Kit-8) assay
The cell counting kit-8 (CCK-8) was used to evaluate cell proliferation. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt], a highly water-soluble tetrazolium salt, is reduced by dehydrogenase activities in cells to give a yellow-color formazan dye, which is soluble in the tissue culture media. The amount of the formazan dye, generated by the activities of dehydrogenases in cells, is directly proportional to the number of living cells. HSC-2 cells were incubated with different Ti surfaces and TCPS controls for 3 h, 6 h, and 24 h. Thereafter 50 μl of CCK-8 (Cell Counting Kit-8; Dojindo, Japan) were added into each well and incubated at 37 °C for 4 h. The optical density (OD) at 450 nm was determined using an ELISA Reader (Molecular Devices, USA) to measure cell growth rate.
2.5
MTT assay
HSC-2 cells were incubated with different Ti surfaces and TCPS controls for 48 h and 72 h. Then the MTT assays were performed. In brief, MTT solution [5 mg/ml, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide in PBS] (Sigma, Germany) was added to each well and culture plates were incubated at 37 °C for 4 h. The medium was removed and 500 μl dimethylsulfoxide (DMSO) was added to each well, then followed by 5 min incubation on a shaker. Finally, the optical density was measured at 550 nm with an ELISA Reader (Molecular Devices, USA).
2.6
Time-lapse microscopy
This technique was applied to monitor HSC-2 behavior on various titanium surfaces. HSC-2 were harvested and thereafter incubated with 2.5 μM CellTracker™ Orange CMRA (Molecular Probes™, Invitrogen, UK, Molecular Probes, Eugene, OR, USA) for 30 min. Then the cell tracker solution was replaced with fresh medium and the cultures were incubated for another 30 min at 37 °C. After centrifugation with 1280 rpm for 6 min, cells were plated at a seeding density of 1 × 10 5 cells per well. HSC-2 were grown on different titanium surfaces and TCPS controls in 4-well cell culture plates (Nunclon, Roskilde, Denmark) and were investigated with fluorescence microscopy using an upright Nikon microscope (Nikon Eclipse E 800 M microscope; Nikon Instruments Europe B.V., Badhoevedorp, Netherlands) equipped with a motorized stage (Prior, Cambridge, Great Britain). For this purpose the microscope has been equipped with an individually designed incubator box in order to provide an atmosphere of 37 °C and 5% CO 2 . Fluorescently labeled HSC-2 cells were photographed every 30 min for more than 168 h with a Roper Scientific digital camera Cascade:512F (Photometrics, Tucson, AZ, USA). Two specimens of each titanium surface were investigated by time-lapse microscopy. Pictures were entered in a database and combined to time-lapse movies after the termination of the experiments (data not shown).
2.7
RNA isolation and real-time QPCR assay
The Ti disks were placed in 24-well tissue culture plates (TPP, Swizerland). HSC-2 cultures were incubated with different Ti surfaces and TCPS controls at a seeding density of 1 × 10 5 cells/well for 120 h and were repeated three times . For RNA extraction at least 4 wells per type of implant or control were used in every experiment. Total cellular RNA was extracted from cells using TRI Reagent (Ambion, Applied Biosystem, Austria) according to the manufacturer’s protocol. RNA concentration was measured at A260 with Biophotometer (Eppendorf AG, Germany). Aliquots of 2 μg total RNA were converted into cDNA by using a kit (High capacity cDNA transcription kit, Applied Biosystem, Austria) following the instructions of the manufacturer. CKs are basal cell markers in human stratified squamous epithelia, the integrin α6β4 is considered to form tight adhesive junctions between cells and the basement membrane. Focal contacts can be identified by the presence of the actin binding protein vinculin. TGF-β, regulating cell growth and differentiation, is expressed in several cell types including epithelial cells. For CK14, vinculin, integrin α6, integrin β4, TGF-β1, TGF-β3 and TGF-β genes expression analyses by quantitative real-time PCR was achieved by means of the ABI PRISM 7000 Sequence Detection System (Applied Biosystem, USA). The housekeeping gene β-actin was used as endogeneous control for target gene expression evaluation. Quantitative real-time PCR was performed using the Taqman Gene Expression Assay (Applied Biosystem, Austria). The probes and the primers were synthesized by Applied Biosystems (assay IDs integrin α6: Hs00173952_m1, integrin β4: Hs00173995_m1, CK14: Hs00559328_m1, vinculin: Hs00243320_m1, TGF-β: Hs99999918_m1, TGF-β1: Hs00171257_m1, TGF-β3: Hs00234245_m1 and β-actin: Hs99999903_m1) and the amplification reactions were performed with Taqman qPCR Master Mix (Applied Biosystem, Austria). Briefly, 25 ng of reverse transcribed cDNA template were applied for the amplification reaction. The amplification was initially incubated at 50 °C for 2 min, then 95 °C for 10 min and followed by performing 40 cycles for 15 s at 95 °C, 1 min at 60 °C. Triplicate Q-PCR reactions were prepared for each sample. The point at which the PCR product is first detected above a fixed threshold, termed cycle threshold (Ct), was determined for each sample. Data of samples were presented by calculating the comparative Ct with the formula 2 (−ΔΔCt) , which stands for the difference of Ct between a gene of interest and the housekeeping gene β-actin for one sample compared to a calibrator which is the TCPS control in this study.
2.8
Statistical analysis
Three replicate experiments were performed. Data were presented as mean ± standard error of the mean (SEM) and a one-way analysis of variance (ANOVA) with Tukey HSD tests was performed to assess the significance. Values of p < 0.05 were considered to be statistically significant. All data analyses were performed using the SPSS 14.0 software.
2
Materials and methods
2.1
Preparation of titanium disks
Commercially pure Ti disks (grade 2, Institut Straumann AG, Basel, Switzerland) were characterized by cylindrical shape with a diameter of 15 mm and 1 mm in thickness.
Surface chemistry is a variable for peri-implant healing since it influences surface energy and wettability and therefore the behavior of epithelial cells . Increased wettability enhances interaction between the implant surface and the biologic environment . More recently, an advanced technology of improving surface energy and wettability has been created to modify surface chemistry of Ti implants . Surface roughness is an other variable influencing the behavior of epithelial cells .
In this study, four kinds of surfaces were used: hydrophobic acid-etched (A) surface, modified [hydrophilic] acid-etched (modA) surface, hydrophobic coarse-grit-blasted and acid-etched (SLA) surface and modified [hydrophilic] coarse-grit-blasted and acid-etched (modSLA) surface. The preparation of Ti disks has been described previously . The chemical composition of all surfaces was determined by X-ray photoelectron spectroscopy whereas surface topography and roughness were characterized using scanning electron microscopy and white light confocal microscope, respectively. Hydrophilicity and contact angle hysteresis were tensiometriclly examined by the Wilhelmy method by means of an electrobalance .
2.2
Cell culture
HSC-2 (Oral squamous cell carcinoma cell line, Japan Health Sciences Foundation, Health Science Research Resources Bank, JCRBD 6222) cells were used as a model to investigate the effects of surface and material variations on epithelial cells. The HSC-2 cultures were maintained in MEM medium (Gibco, Austria), supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 10% FCS. Cultures were subdivided by using Trypsin–EDTA solution (1:250, PAA, Austria). All experiments were performed using cells between fifth and eighth passage. The culture medium was changed every 2 days.
2.3
Cell proliferation
The Ti disks were placed in 24-well tissue culture plates (TPP, Switzerland) and then 1 × 10 5 cells were added to each well and were incubated at 37 °C and 5% CO 2 in a humidified atmosphere. In every experiment, six wells with each of the four types of Ti disks and six wells with no disks (tissue culture polystyrene [TCPS] controls) were used. All subsequent experiments were repeated three times.
2.4
CCK-8 (Cell Counting Kit-8) assay
The cell counting kit-8 (CCK-8) was used to evaluate cell proliferation. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt], a highly water-soluble tetrazolium salt, is reduced by dehydrogenase activities in cells to give a yellow-color formazan dye, which is soluble in the tissue culture media. The amount of the formazan dye, generated by the activities of dehydrogenases in cells, is directly proportional to the number of living cells. HSC-2 cells were incubated with different Ti surfaces and TCPS controls for 3 h, 6 h, and 24 h. Thereafter 50 μl of CCK-8 (Cell Counting Kit-8; Dojindo, Japan) were added into each well and incubated at 37 °C for 4 h. The optical density (OD) at 450 nm was determined using an ELISA Reader (Molecular Devices, USA) to measure cell growth rate.
2.5
MTT assay
HSC-2 cells were incubated with different Ti surfaces and TCPS controls for 48 h and 72 h. Then the MTT assays were performed. In brief, MTT solution [5 mg/ml, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide in PBS] (Sigma, Germany) was added to each well and culture plates were incubated at 37 °C for 4 h. The medium was removed and 500 μl dimethylsulfoxide (DMSO) was added to each well, then followed by 5 min incubation on a shaker. Finally, the optical density was measured at 550 nm with an ELISA Reader (Molecular Devices, USA).
2.6
Time-lapse microscopy
This technique was applied to monitor HSC-2 behavior on various titanium surfaces. HSC-2 were harvested and thereafter incubated with 2.5 μM CellTracker™ Orange CMRA (Molecular Probes™, Invitrogen, UK, Molecular Probes, Eugene, OR, USA) for 30 min. Then the cell tracker solution was replaced with fresh medium and the cultures were incubated for another 30 min at 37 °C. After centrifugation with 1280 rpm for 6 min, cells were plated at a seeding density of 1 × 10 5 cells per well. HSC-2 were grown on different titanium surfaces and TCPS controls in 4-well cell culture plates (Nunclon, Roskilde, Denmark) and were investigated with fluorescence microscopy using an upright Nikon microscope (Nikon Eclipse E 800 M microscope; Nikon Instruments Europe B.V., Badhoevedorp, Netherlands) equipped with a motorized stage (Prior, Cambridge, Great Britain). For this purpose the microscope has been equipped with an individually designed incubator box in order to provide an atmosphere of 37 °C and 5% CO 2 . Fluorescently labeled HSC-2 cells were photographed every 30 min for more than 168 h with a Roper Scientific digital camera Cascade:512F (Photometrics, Tucson, AZ, USA). Two specimens of each titanium surface were investigated by time-lapse microscopy. Pictures were entered in a database and combined to time-lapse movies after the termination of the experiments (data not shown).
2.7
RNA isolation and real-time QPCR assay
The Ti disks were placed in 24-well tissue culture plates (TPP, Swizerland). HSC-2 cultures were incubated with different Ti surfaces and TCPS controls at a seeding density of 1 × 10 5 cells/well for 120 h and were repeated three times . For RNA extraction at least 4 wells per type of implant or control were used in every experiment. Total cellular RNA was extracted from cells using TRI Reagent (Ambion, Applied Biosystem, Austria) according to the manufacturer’s protocol. RNA concentration was measured at A260 with Biophotometer (Eppendorf AG, Germany). Aliquots of 2 μg total RNA were converted into cDNA by using a kit (High capacity cDNA transcription kit, Applied Biosystem, Austria) following the instructions of the manufacturer. CKs are basal cell markers in human stratified squamous epithelia, the integrin α6β4 is considered to form tight adhesive junctions between cells and the basement membrane. Focal contacts can be identified by the presence of the actin binding protein vinculin. TGF-β, regulating cell growth and differentiation, is expressed in several cell types including epithelial cells. For CK14, vinculin, integrin α6, integrin β4, TGF-β1, TGF-β3 and TGF-β genes expression analyses by quantitative real-time PCR was achieved by means of the ABI PRISM 7000 Sequence Detection System (Applied Biosystem, USA). The housekeeping gene β-actin was used as endogeneous control for target gene expression evaluation. Quantitative real-time PCR was performed using the Taqman Gene Expression Assay (Applied Biosystem, Austria). The probes and the primers were synthesized by Applied Biosystems (assay IDs integrin α6: Hs00173952_m1, integrin β4: Hs00173995_m1, CK14: Hs00559328_m1, vinculin: Hs00243320_m1, TGF-β: Hs99999918_m1, TGF-β1: Hs00171257_m1, TGF-β3: Hs00234245_m1 and β-actin: Hs99999903_m1) and the amplification reactions were performed with Taqman qPCR Master Mix (Applied Biosystem, Austria). Briefly, 25 ng of reverse transcribed cDNA template were applied for the amplification reaction. The amplification was initially incubated at 50 °C for 2 min, then 95 °C for 10 min and followed by performing 40 cycles for 15 s at 95 °C, 1 min at 60 °C. Triplicate Q-PCR reactions were prepared for each sample. The point at which the PCR product is first detected above a fixed threshold, termed cycle threshold (Ct), was determined for each sample. Data of samples were presented by calculating the comparative Ct with the formula 2 (−ΔΔCt) , which stands for the difference of Ct between a gene of interest and the housekeeping gene β-actin for one sample compared to a calibrator which is the TCPS control in this study.
2.8
Statistical analysis
Three replicate experiments were performed. Data were presented as mean ± standard error of the mean (SEM) and a one-way analysis of variance (ANOVA) with Tukey HSD tests was performed to assess the significance. Values of p < 0.05 were considered to be statistically significant. All data analyses were performed using the SPSS 14.0 software.
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