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Response of human fibroblasts to implant surface coated with titanium dioxide photocatalytic films
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Journal of Prosthodontic Research 54 (2010) 185–191 www.elsevier.com/locate/jpor
Original article
Response of human fibroblasts to implant surface coated with titanium dioxide photocatalytic films Noriyuki Hoshi DDSa,*, Hideyuki Negishi PhDb, Shusaku Okada DDS, PhDc, Toru Nonami PhDd, Katsuhiko Kimoto DDS, PhDa a
Division of Fixed Prosthodontics, Department of Oral & Maxillofacial Rehabilitation, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka, Kanagawa 238-8580, Japan b Division of Dental Bioengineering, Kanagawa Dental College, Yokosuka, Kanagawa, Japan c Department of Oral Medicine Division of Restorative Dentistry, Kanagawa Dental College, Yokosuka, Kanagawa, Japan d School of Life Science and Technology, Chukyo University, Toyoda, Aichi, Japan Received 8 December 2009; received in revised form 2 April 2010; accepted 16 April 2010 Available online 15 May 2010
Abstract Purpose: This study was to develop a titanium dioxide (TiO2)-coated implant abutment, surface with ultraviolet (UV) light-induced hydrophilicity and investigate the initial response of human, fibroblasts to the surface modification. Materials and methods: Commercially pure titanium (JIS 2 grade) disks were coated with TiO2 to various, thicknesses (1, 2 or 3 mm) using peroxotitanium acid solution. The surface characteristics of each disk, were examined with X-ray diffraction (XRD), surface roughness equipment and scanning electron, microscopy (SEM). The hydrophilic change of each disk was determined by the contact angles at 0–24 h, after 24-h UV irradiation. The biological response at the surface of each disk was examined by using, human periodontal ligament fibroblasts (HPLFs). The data were statistically analyzed with analysis of variance (ANOVA) and multiple-comparison tests. Results: The TiO2-coated disk surface had an anatase structure. Surface roughness did not differ, significantly among the disks; the surface morphology was smooth and had a hydrophilic or superhydrophilic, status. HPLF proliferation significantly increased on the TiO2-coated disks compared with the uncoated disks and depended upon the coated film thickness. Conclusion: An anatase TiO2-coated surface under UV irradiation markedly improves the initial response of human fibroblasts. # 2010 Japan Prosthodontic Society. Published by Elsevier Ireland. All rights reserved. Keywords: Dental implant abutment; Titanium dioxide; Photocatalyst; Fibroblast; Peroxotitanium acid solution
1. Introduction Mucosal peri-implant tissue is the transmucosal part and is always exposed to the risk of infection from the oral environment. Mucosal infections lead to inflammatory reactions in the mucosal tissue surrounding dental implant surfaces and subsequently induce loss of bone supporting the implant [1,2]. In general, mucosal tissue consists of an epithelial component and a connective tissue component; mucosal peri-implant tissue has some features in common with gingival tissue of the natural dentition [3–5]. There is however an important difference between these two types of mucosal tissue.
* Corresponding author. Tel.: +81 468 22 8861; fax: +81 468 22 8861. E-mail address: [email protected] (N. Hoshi).
The connective tissue attachment in the natural dentition is characterized by gingival collagen fiber bundles running perpendicular to root cementum, whereas an implant surface lacks root cementum and the orientation of the collagen fibers is parallel or circumferential to the implant surface [6,7]. Furthermore, in comparison with the connective tissue of the natural dentition, the connective tissue around an implant is characterized by a scar-like structure that is rich in collagen but deficient in fibroblast rich [8]. Fibroblastic richness of the mucosal tissue surrounding dental implant surfaces ensures high turnover and may play a role in establishing and maintaining a proper mucosal seal and protective mechanism [9–11]. For treatment success, it is therefore important to develop bioactive materials that allow easy fibroblastic proliferation in mucosal peri-implant tissue. Anatase titanium dioxide (TiO2) forms photocatalytic activity induced by ultraviolet (UV) irradiation [12], and has
1883-1958/$ – see front matter # 2010 Japan Prosthodontic Society. Published by Elsevier Ireland. All rights reserved. doi:10.1016/j.jpor.2010.04.005
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recently been applied to develop antifungal, stain-proof and self-cleaning materials in dentistry for various photocatalytic effects [13–16]. In particular, one of the unique characteristics of TiO2-coated surfaces is the microstructural composition of the hydrophilic and oleophilic phases under UV irradiation [17]. Interestingly, a recent study has demonstrated that this hydrophilic status enhances the attachment, spread, proliferation and differentiation of osteoblasts and mesenchymal stem cells; furthermore, it promotes protein adsorption and bone formation [16,18–21]. In previous observations of the mucosal tissue response to the implant surface, surface modification of titanium (Ti) abutment materials significantly influenced the attachment and growth of fibroblasts [22–26]. We therefore hypothesized that an UV light-induced hydrophilic surface of TiO2 film enhances the biological response of fibroblasts. In this study, we aimed to develop a new abutment surface coated with TiO2 film and having UV light-induced hydrophilicity, and investigate the initial response of human fibroblasts to this surface modification.
specimens were used for surface roughness determination. The centerline average roughness value (Ra) was determined by averaging the values of three random areas per disk. Each determination was carried out at a length of 0.400 mm, determination speed of 0.300 mm/s and cut-off wavelength of 0.080 mm.
2. Materials and methods
2.4. Cell culture
2.1. Specimen preparation
Human periodontal ligament was collected from the extracted teeth (first premolars) of 14–20-year-old patients undergoing orthodontic treatment at the hospital dental office. According to the guidelines of the ethics committee (Ethic Committee of Kanagawa Dental College), we explained the profile of our study to the patients and collected the samples after obtaining written consent. The periodontal ligament of the central part of the roots was gently exfoliated with a scalpel and placed into wells of a 24-multiwell plate (FALCON 3043; Becton, Dickinson and Company, New Jersey, USA). Plastic sheets for tissue culture (Wako Pure Chemical Industries, Osaka, Japan) were placed on the tissue samples, pressed and fixed to create the primary culture [28]. Dulbecco’s modified Eagle’s medium (Gibco, New Jersey, USA) with 5% foetal bovine serum (Thermo Trace, Noble Park, Australia), 0.25 mM L-ascorbic acid-2-phosphate (Wako Pure Chemical Industries, Osaka, Japan), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco, New Jersey, USA) was used as the culture medium and incubated in 5% CO2 and 95% air with a humectation gas phase at 37 8C. Cellular migration from the tissue was confirmed after 3 or 4 days, followed by change of the culture medium. The cells that migrated from the tissue about 2 weeks later proliferated and reached about 80% confluence, which was confirmed by phase-contrast microscopy. They were treated in Ca2+-, Mg2+-free phosphate-buffered saline (Nippon Seiyaku, Tokyo, Japan) including 0.25% trypsin (Difco Laboratories, New Jersey, USA) and subcultured. The cells from passages 6–8 were used for the experiments.
Commercially pure Ti (JIS 2 grade) disks of 45 mm diameter and 0.8 mm thick were used as substrates. The surfaces of each Ti disk were polished with silicon carbide paper (#1200 grit) under running tap water, and then coated with peroxotitanium acid solution (TTC-40A; Takahara Corp., Tokyo, Japan) from a distance of 20 cm with an atomized, followed by air-drying at 25 (normal temperature), 550 and 800 8C [27]. Results described below, without affecting the base materials 25 8C air-dried specimens were randomly divided into four groups as follows: (1) no coating as a control (Cont-Ti), (2) 1-mm-thick coating film (TiO2-1), (3) 2-mm-thick coating film (TiO2-2) and (4) 3-mm-thick coating film (TiO2-3). In addition, the surfaces of glass disks of 45 mm diameter and 0.8 mm thick were coated by using the same procedure (at 25 8C air-dried) and divided into the following four groups: (5) an uncoated glass disk as control (Cont-G), (6) 1-mm-thick coating film (G-TiO2-1), (7) 2-mm-thick coating film (G-TiO2-2) and (8) 3-mm-thick coating film (G-TiO2-3). The thickness of each coating film was confirmed by scanning electron microscopy (SEM; SUPERSCAN SS-550; Shimadzu, Kyoto, Japan) at 15.0 kV after having cut ten disks each of the eight types of specimens. 2.2. Surface characterisation The following analytical methods were used to characterize the TiO2 coating films. Compositional analysis of the TiO2 coating films was conducted by powder X-ray diffraction (XRD; MiniFlex; Rigaku Industrial Corp., Osaka, Japan). The surface morphology of the films was examined by SEM at 15.0 kV. The surface roughness of each coated disk was analyzed by contact surface roughness testing (Surfcom 590 A; Seimitsu, Tokyo, Japan). Three disks each of the eight types of
2.3. Contact angle measurement The hydrophilicity of the coated disks was assessed by measuring the contact angle of 1.5 ml water droplets on the disk surface using a FACE automatic dynamic contact angle device (DCA-VZ type; Kyouwakaimenkagaku, Saitama, Japan). Eight disks each of the eight types of specimens were irradiated with UV light wavelength 368 nm and illumination 3.8 W (FPL27BLB27W; Sankyo Electricity, Osaka, Japan) from a distance of 20 cm for 24 h covered with a box. The contact angles were advancing measured after 0–10 h and 24 h. Further, the side view of water spread onto the specimens was digitally photographed for presentation.
2.5. Observation of cell attachment and proliferation After the 24 h UV irradiation, the disks each of the eight types of specimens were placed centrally in a 60 mm hydrophobic culture dish. Human periodontal ligament
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fibroblasts (HPLFs) were seeded onto each disk at a density of 1.8 104 cells/cm2. The culture medium was renewed every 2 days in a humidified atmosphere of 95% air and 5% CO2 at 37 8C. For eliminating the non-adhesive cells, the specimens of all groups were transferred to a new hydrophobic culture dish, and new medium was poured. After 3 day culture (groups 1–8) and 7 day culture (groups 1–4), the specimens were again transferred to a new hydrophobic culture dish, and the cells were detached by using 3 ml of 0.25% trypsin for 10 min at 37 8C. A haematocytometer (4011; Becton, Dickinson and Company, New Jersey, USA) was used to count the number of detached cells and the affirmation of cell removed on the surface of each specimen used SEM. Furthermore, initial cell behavior of the fibroblasts (spreading and morphology) after 12 h and 3 days of incubation was observed by inverted phase-contrast microscopy (TMS-F 20100; Nikon Corp., Tokyo, Japan). 2.6. Statistical analysis The data are presented as the mean and standard deviation (SD). Statistical analysis was performed by using one-way analysis of variance (ANOVA). When ANOVA indicated a significant difference among the groups, the difference of each group was analyzed by multiple-comparison tests (Scheffe’s test). A P-value less than 0.05 was considered to be statistically significant. 3. Results 3.1. Surface characterisation 3.1.1. XRD pattern The XRD patterns of the 25 8C (normal temperature), 550 8C and 800 8C air-dried TiO2 coating films are shown in Fig. 1. The surfaces of the TiO2 coating films under the drying conditions of 25 8C and 550 8C were detected as the anatase type, whereas that air-dried at 800 8C partly changed to the rutile type. 3.1.2. SEM images Surface micrographs of the coated disk specimens were shown in Fig. 2A. All surfaces revealed a similar surface texture (i.e., with smoothness), and no difference was observable among the groups. 3.1.3. Surface roughness The results of the surface roughness testing were shown in Fig. 2B. The surface roughness ranged from 228.3 22.1 nm to 275.7 23.5 nm. The values were not found to be significantly different between the control and TiO2-coated disks irrespective of the film thickness.
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Fig. 1. The XRD patterns of the TiO2 coating films under different treatment conditions. (A) 25 8C (normal temperature) air-dried TiO2 coating film, (B) 550 8C air-dried TiO2 coating film, (C) 800 8C air-dried TiO2 coating film and (D) rutile TiO2. The surface of the TiO2 coating films under the drying conditions of 25 8C and 550 8C were detected as the anatase type.
UV irradiation, and there was no diachronic change. The TiO2coated surfaces at each film thickness showed a hydrophilic (contact angle < 308) or super-hydrophilic state (contact angle < 58) with and without UV irradiation [29]. All thicknesses of the TiO2 coating films UV-irradiated for 24 h showed extensive hydrophilicity, which was maintained even after 24 h. As for the hydrophilic intensity and durability, the coating disks were more effective than the uncoated disk, and the thick coating film (TiO2-3) were more effective than the thin coating film (TiO2-1) (Fig. 3). Similar tendencies were observed for the glass disks. The hydrophilic intensity and durability, the coating films were more effective than uncoated disk (Fig. 4). 3.2. Cell attachment and proliferation The observations of cellular proliferation on the TiO2-coated surfaces after 12 h and 3 days of incubation are shown in Fig. 5. At 12 h, initial cellular attachment on the disk surface was seen (Fig. 5A), and cellular proliferation became conspicuous (Fig. 5B, C and D). After 3 days, the cells formed cellular bridges and proliferated (Fig. 5E and I). Sufficient cellular proliferation and cellular bridges were observed on the TiO2coated disks (Fig. 5F, G, H, J, K and L). The numbers of HPLFs on the glass and Ti disk surface after culture for 3 days were shown in Fig. 6 and on the Ti disk at 7 days culture were shown in Fig. 7 respectively. Statistical analysis showed that the number of cells on the TiO2-coated disks was significantly higher than that on the uncoated disks. Further, the number of cells on the thick coating films was significantly higher than that on the thin coating film. The cellular response on the disk surface was dependant on the coated film thickness. 4. Discussion
3.1.4. Contact angle measurement The contact angle measurements for the Ti disks and glass disks were shown in Figs. 3 and 4, respectively. The contact angle of Cont-Ti showed hydrophobicity both with and without
Regarding TiO2 photocatalysts, it is well known that oxidative decomposition promoted by oxidation power of a positive hole generated by photoexcitation [12]. Thereafter,
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Fig. 2. Surface characterisation of the TiO2 coating film. (A) SEM imaging of the surface; scale bar = 100 mm. (B) Surface roughness measurement; the data are the mean SD for all disks (n = 9). Cont-Ti: no coating (control); TiO2-1: 1-mm-thick coating film; TiO2-2: 2-mm-thick coating film; TiO2-3: 3-mm-thick coating film. All surface revealed a similar picture, and no difference was observable among the group. The values were not found to be significantly different between the control and TiO2-coated disks irrespective of the film thickness.
Fig. 3. Time-dependent contact angle of the TiO2-coated surface (Ti disks). The data are the mean SD for all disks (n = 8.). Cont-Ti: no coating (control); TiO2-1: 1-mm-thick coating film; TiO2-2: 2-mm-thick coating film; TiO2-3: 3mm-thick coating film. Shown a picture of the state of the water to drip into the bottom. *All data intervals were significantly different ( p < 0.05). As for the hydrophilic intensity and durability, the TiO2-coated disks were more effective than the uncoated disks, and the thick coating film (TiO2-3) were more effective than the thin coating film (TiO2-1).
Fig. 4. Time-dependent contact angle of the TiO2-coated surface (glass disks). The data are the mean SD for all disks (n = 8). Cont-G: no coating (control); G-TiO2-1: 1-mm-thick coating film; G-TiO2-2: 2-mm-thick coating film; GTiO2-3: 3-mm-thick coating film. Shown a picture of the state of the water to drip into the bottom. *All data intervals were significantly different ( p < 0.05). As for the hydrophilic intensity and durability, the TiO2-coated disks were more effective than the uncoated disks.
Wang et al. [17] found that TiO2 surfaces under UV irradiation produce a hydrophilic surface. After further studies, the mechanism of hydrophilicity development was explained by decomposition and removal of organic compounds by a photogenerated positive hole and hydrophilic domain formation by increase in surface hydroxyl groups [30–32]. At present, TiO2 photocatalysts are used in various industries because of these phenomenal characteristics. To obtain the best characteristics, a TiO2 photocatalyst must be coated on base materials and immobilized without structural change of the base materials. Wet and dry processes can be used to achieve such immobilization. The former requires application of
solvents as a raw material of TiO2 before sintering, whereas the latter can be conducted without application of solvents. In both processes, however, heat application, adhesive layers and special equipments are needed. We used a peroxotitanium solution developed for application to any surface without special equipments and solvents for coating TiO2. This neutral solution can be applied as a translucent coating film without heat application at room temperature [27]. Moreover, as shown in Fig. 2, the coated surface was highly smooth. We confirmed that coating with a thickness at the micrometer level is possible. From these findings, we believe that this technique can provide photo-
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Fig. 5. Representative photographs of HPLF proliferation on the TiO2 coating film (glass disks) at 12 h and 3 days after seeding. Scale bar = 100 mm. Cont-G: no coating (control); G-TiO2-1: 1-mm-thick coating film; G-TiO2-2: 2-mm-thick coating film; G-TiO2-3: 3-mm-thick coating film. At 12 h, initial cellular attachment on the disk surface was seen (A), and cellular proliferation became conspicuous (B–D). After 3 days, the cells formed cellular bridges and proliferation (E and I). Sufficient cellular proliferation and cellular bridges were observed on the TiO2-coated disks (F, G, H, J, K, and L). Arrows indicate the cellular bridges.
Fig. 6. Examination of HPLF proliferation on the TiO2 coating film. (A) TiO2-coated surface at 3 days after seeding (glass disks). (B) TiO2-coated surface at 3 days after seeding (Ti disks). All data are expressed as the mean SD (n = 9 for both types of disks). *All data intervals were significantly different ( p < 0.05). The number of cells on the TiO2-coated disks was higher than that on the uncoated disks. The cellular response on the disk surface was dependant on the coated film thickness.
catalytic effects without affecting the base material surface structure of commercial implants. Regarding the hydrophilic status, as can be seen in Figs. 3 and 4, the same level of slight hydrophilicity was observed at
the different film thicknesses even without UV irradiation. Under UV irradiation, sufficient hydrophilicity was obtained even with thin application of the TiO2 photocatalyst; interestingly, we found that the thick coating films could
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structured water. On that surface, after attachment of plasma glycoproteins, extracellular matrix proteins such as collagen I and fibronectin are attached through various ions. These extracellular matrix proteins are critical for regulating cellular function such as cell adhesion, shape and migration [36,37]. We speculate that after this series of surface layering, fibroblasts finally attached onto the TiO2 surface. Consequently, the thick coating film maintaining hydrophilicity for a long time works effectively on the fibroblasts because of its long-lasting effect on their growth. Therefore, such surface modification with hydrophilicity may promote the formation of implant-circumferential connective tissue rich in fibroblasts and allow acquisition of a wellfunctioning barrier mechanism against the external environment. Application of TiO2 photocatalysts with mucosal peri-implant tissue is of great interest at present and will need further study. 5. Conclusion We consider that the newly developed anatase-TiO2 coating film under UV irradiation markedly improves the conductive response of fibroblasts. Fig. 7. Examination of HPLF proliferation on the TiO2 coating film. TiO2coated surface at 7 days after seeding (Ti disks). All data are expressed as the mean SD (n = 9). *All data intervals were significantly different ( p < 0.05). The number of cells on the TiO2-coated disks was higher than that on the uncoated disks. The cellular response on the disk surface was dependant on the coated film thickness.
retain surface hydrophilicity for a long period. Previous studies have reported that this longevity is established by threedimensional arrangements of the anatase crystal structure with stomata of TiO2 [30,33]. Namely, the hydrophilicity of TiO2 surfaces is by nature relatively low, but with UV irradiation, oxygen deletion-promoting dissociative adsorption occurs on the surface, and then, the surface structure changes with formation of hydrophilic domains accompanied with increase of surface hydroxyl groups. Hence, we suggest that the hydrophilicity following the photocatalytic reaction by UV irradiation is determined by three-dimensional arrangements of the anatase crystal structure included in the coating film and that the thick and high-volume membrane has a larger surface area of hydrophilic domains, resulting in higher hydrophilicity. The existence of a hydrophilic status is advantageous to improve cellular attachment and proliferation. In fact, as can be seen in Figs. 5–7, the HPLFs showed an increasing tendency on the TiO2-coated surface compared with the uncoated disks. Especially, the thick TiO2 film rendered a prominent increasing tendency. The mechanism can be explained as follows. In addition to formation of the hydrophilic domains, obtainment of fresh surfaces due to surface hydrocarbon removal by the photocatalytic reaction of TiO2 [29] and resolution of the organic contents result in higher rates of surface hydroxyl groups on the thick film than the thin film [34,35]. Specifically, because the thick films have high hydrophilicity and surface energy, water is attached first on the surface to form a layer of
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Journal of Prosthodontic Research 54 (2010) 185–191 www.elsevier.com/locate/jpor
Original article
Response of human fibroblasts to implant surface coated with titanium dioxide photocatalytic films Noriyuki Hoshi DDSa,*, Hideyuki Negishi PhDb, Shusaku Okada DDS, PhDc, Toru Nonami PhDd, Katsuhiko Kimoto DDS, PhDa a
Division of Fixed Prosthodontics, Department of Oral & Maxillofacial Rehabilitation, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka, Kanagawa 238-8580, Japan b Division of Dental Bioengineering, Kanagawa Dental College, Yokosuka, Kanagawa, Japan c Department of Oral Medicine Division of Restorative Dentistry, Kanagawa Dental College, Yokosuka, Kanagawa, Japan d School of Life Science and Technology, Chukyo University, Toyoda, Aichi, Japan Received 8 December 2009; received in revised form 2 April 2010; accepted 16 April 2010 Available online 15 May 2010
Abstract Purpose: This study was to develop a titanium dioxide (TiO2)-coated implant abutment, surface with ultraviolet (UV) light-induced hydrophilicity and investigate the initial response of human, fibroblasts to the surface modification. Materials and methods: Commercially pure titanium (JIS 2 grade) disks were coated with TiO2 to various, thicknesses (1, 2 or 3 mm) using peroxotitanium acid solution. The surface characteristics of each disk, were examined with X-ray diffraction (XRD), surface roughness equipment and scanning electron, microscopy (SEM). The hydrophilic change of each disk was determined by the contact angles at 0–24 h, after 24-h UV irradiation. The biological response at the surface of each disk was examined by using, human periodontal ligament fibroblasts (HPLFs). The data were statistically analyzed with analysis of variance (ANOVA) and multiple-comparison tests. Results: The TiO2-coated disk surface had an anatase structure. Surface roughness did not differ, significantly among the disks; the surface morphology was smooth and had a hydrophilic or superhydrophilic, status. HPLF proliferation significantly increased on the TiO2-coated disks compared with the uncoated disks and depended upon the coated film thickness. Conclusion: An anatase TiO2-coated surface under UV irradiation markedly improves the initial response of human fibroblasts. # 2010 Japan Prosthodontic Society. Published by Elsevier Ireland. All rights reserved. Keywords: Dental implant abutment; Titanium dioxide; Photocatalyst; Fibroblast; Peroxotitanium acid solution
1. Introduction Mucosal peri-implant tissue is the transmucosal part and is always exposed to the risk of infection from the oral environment. Mucosal infections lead to inflammatory reactions in the mucosal tissue surrounding dental implant surfaces and subsequently induce loss of bone supporting the implant [1,2]. In general, mucosal tissue consists of an epithelial component and a connective tissue component; mucosal peri-implant tissue has some features in common with gingival tissue of the natural dentition [3–5]. There is however an important difference between these two types of mucosal tissue.
* Corresponding author. Tel.: +81 468 22 8861; fax: +81 468 22 8861. E-mail address: [email protected] (N. Hoshi).
The connective tissue attachment in the natural dentition is characterized by gingival collagen fiber bundles running perpendicular to root cementum, whereas an implant surface lacks root cementum and the orientation of the collagen fibers is parallel or circumferential to the implant surface [6,7]. Furthermore, in comparison with the connective tissue of the natural dentition, the connective tissue around an implant is characterized by a scar-like structure that is rich in collagen but deficient in fibroblast rich [8]. Fibroblastic richness of the mucosal tissue surrounding dental implant surfaces ensures high turnover and may play a role in establishing and maintaining a proper mucosal seal and protective mechanism [9–11]. For treatment success, it is therefore important to develop bioactive materials that allow easy fibroblastic proliferation in mucosal peri-implant tissue. Anatase titanium dioxide (TiO2) forms photocatalytic activity induced by ultraviolet (UV) irradiation [12], and has
1883-1958/$ – see front matter # 2010 Japan Prosthodontic Society. Published by Elsevier Ireland. All rights reserved. doi:10.1016/j.jpor.2010.04.005
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recently been applied to develop antifungal, stain-proof and self-cleaning materials in dentistry for various photocatalytic effects [13–16]. In particular, one of the unique characteristics of TiO2-coated surfaces is the microstructural composition of the hydrophilic and oleophilic phases under UV irradiation [17]. Interestingly, a recent study has demonstrated that this hydrophilic status enhances the attachment, spread, proliferation and differentiation of osteoblasts and mesenchymal stem cells; furthermore, it promotes protein adsorption and bone formation [16,18–21]. In previous observations of the mucosal tissue response to the implant surface, surface modification of titanium (Ti) abutment materials significantly influenced the attachment and growth of fibroblasts [22–26]. We therefore hypothesized that an UV light-induced hydrophilic surface of TiO2 film enhances the biological response of fibroblasts. In this study, we aimed to develop a new abutment surface coated with TiO2 film and having UV light-induced hydrophilicity, and investigate the initial response of human fibroblasts to this surface modification.
specimens were used for surface roughness determination. The centerline average roughness value (Ra) was determined by averaging the values of three random areas per disk. Each determination was carried out at a length of 0.400 mm, determination speed of 0.300 mm/s and cut-off wavelength of 0.080 mm.
2. Materials and methods
2.4. Cell culture
2.1. Specimen preparation
Human periodontal ligament was collected from the extracted teeth (first premolars) of 14–20-year-old patients undergoing orthodontic treatment at the hospital dental office. According to the guidelines of the ethics committee (Ethic Committee of Kanagawa Dental College), we explained the profile of our study to the patients and collected the samples after obtaining written consent. The periodontal ligament of the central part of the roots was gently exfoliated with a scalpel and placed into wells of a 24-multiwell plate (FALCON 3043; Becton, Dickinson and Company, New Jersey, USA). Plastic sheets for tissue culture (Wako Pure Chemical Industries, Osaka, Japan) were placed on the tissue samples, pressed and fixed to create the primary culture [28]. Dulbecco’s modified Eagle’s medium (Gibco, New Jersey, USA) with 5% foetal bovine serum (Thermo Trace, Noble Park, Australia), 0.25 mM L-ascorbic acid-2-phosphate (Wako Pure Chemical Industries, Osaka, Japan), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco, New Jersey, USA) was used as the culture medium and incubated in 5% CO2 and 95% air with a humectation gas phase at 37 8C. Cellular migration from the tissue was confirmed after 3 or 4 days, followed by change of the culture medium. The cells that migrated from the tissue about 2 weeks later proliferated and reached about 80% confluence, which was confirmed by phase-contrast microscopy. They were treated in Ca2+-, Mg2+-free phosphate-buffered saline (Nippon Seiyaku, Tokyo, Japan) including 0.25% trypsin (Difco Laboratories, New Jersey, USA) and subcultured. The cells from passages 6–8 were used for the experiments.
Commercially pure Ti (JIS 2 grade) disks of 45 mm diameter and 0.8 mm thick were used as substrates. The surfaces of each Ti disk were polished with silicon carbide paper (#1200 grit) under running tap water, and then coated with peroxotitanium acid solution (TTC-40A; Takahara Corp., Tokyo, Japan) from a distance of 20 cm with an atomized, followed by air-drying at 25 (normal temperature), 550 and 800 8C [27]. Results described below, without affecting the base materials 25 8C air-dried specimens were randomly divided into four groups as follows: (1) no coating as a control (Cont-Ti), (2) 1-mm-thick coating film (TiO2-1), (3) 2-mm-thick coating film (TiO2-2) and (4) 3-mm-thick coating film (TiO2-3). In addition, the surfaces of glass disks of 45 mm diameter and 0.8 mm thick were coated by using the same procedure (at 25 8C air-dried) and divided into the following four groups: (5) an uncoated glass disk as control (Cont-G), (6) 1-mm-thick coating film (G-TiO2-1), (7) 2-mm-thick coating film (G-TiO2-2) and (8) 3-mm-thick coating film (G-TiO2-3). The thickness of each coating film was confirmed by scanning electron microscopy (SEM; SUPERSCAN SS-550; Shimadzu, Kyoto, Japan) at 15.0 kV after having cut ten disks each of the eight types of specimens. 2.2. Surface characterisation The following analytical methods were used to characterize the TiO2 coating films. Compositional analysis of the TiO2 coating films was conducted by powder X-ray diffraction (XRD; MiniFlex; Rigaku Industrial Corp., Osaka, Japan). The surface morphology of the films was examined by SEM at 15.0 kV. The surface roughness of each coated disk was analyzed by contact surface roughness testing (Surfcom 590 A; Seimitsu, Tokyo, Japan). Three disks each of the eight types of
2.3. Contact angle measurement The hydrophilicity of the coated disks was assessed by measuring the contact angle of 1.5 ml water droplets on the disk surface using a FACE automatic dynamic contact angle device (DCA-VZ type; Kyouwakaimenkagaku, Saitama, Japan). Eight disks each of the eight types of specimens were irradiated with UV light wavelength 368 nm and illumination 3.8 W (FPL27BLB27W; Sankyo Electricity, Osaka, Japan) from a distance of 20 cm for 24 h covered with a box. The contact angles were advancing measured after 0–10 h and 24 h. Further, the side view of water spread onto the specimens was digitally photographed for presentation.
2.5. Observation of cell attachment and proliferation After the 24 h UV irradiation, the disks each of the eight types of specimens were placed centrally in a 60 mm hydrophobic culture dish. Human periodontal ligament
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fibroblasts (HPLFs) were seeded onto each disk at a density of 1.8 104 cells/cm2. The culture medium was renewed every 2 days in a humidified atmosphere of 95% air and 5% CO2 at 37 8C. For eliminating the non-adhesive cells, the specimens of all groups were transferred to a new hydrophobic culture dish, and new medium was poured. After 3 day culture (groups 1–8) and 7 day culture (groups 1–4), the specimens were again transferred to a new hydrophobic culture dish, and the cells were detached by using 3 ml of 0.25% trypsin for 10 min at 37 8C. A haematocytometer (4011; Becton, Dickinson and Company, New Jersey, USA) was used to count the number of detached cells and the affirmation of cell removed on the surface of each specimen used SEM. Furthermore, initial cell behavior of the fibroblasts (spreading and morphology) after 12 h and 3 days of incubation was observed by inverted phase-contrast microscopy (TMS-F 20100; Nikon Corp., Tokyo, Japan). 2.6. Statistical analysis The data are presented as the mean and standard deviation (SD). Statistical analysis was performed by using one-way analysis of variance (ANOVA). When ANOVA indicated a significant difference among the groups, the difference of each group was analyzed by multiple-comparison tests (Scheffe’s test). A P-value less than 0.05 was considered to be statistically significant. 3. Results 3.1. Surface characterisation 3.1.1. XRD pattern The XRD patterns of the 25 8C (normal temperature), 550 8C and 800 8C air-dried TiO2 coating films are shown in Fig. 1. The surfaces of the TiO2 coating films under the drying conditions of 25 8C and 550 8C were detected as the anatase type, whereas that air-dried at 800 8C partly changed to the rutile type. 3.1.2. SEM images Surface micrographs of the coated disk specimens were shown in Fig. 2A. All surfaces revealed a similar surface texture (i.e., with smoothness), and no difference was observable among the groups. 3.1.3. Surface roughness The results of the surface roughness testing were shown in Fig. 2B. The surface roughness ranged from 228.3 22.1 nm to 275.7 23.5 nm. The values were not found to be significantly different between the control and TiO2-coated disks irrespective of the film thickness.
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Fig. 1. The XRD patterns of the TiO2 coating films under different treatment conditions. (A) 25 8C (normal temperature) air-dried TiO2 coating film, (B) 550 8C air-dried TiO2 coating film, (C) 800 8C air-dried TiO2 coating film and (D) rutile TiO2. The surface of the TiO2 coating films under the drying conditions of 25 8C and 550 8C were detected as the anatase type.
UV irradiation, and there was no diachronic change. The TiO2coated surfaces at each film thickness showed a hydrophilic (contact angle < 308) or super-hydrophilic state (contact angle < 58) with and without UV irradiation [29]. All thicknesses of the TiO2 coating films UV-irradiated for 24 h showed extensive hydrophilicity, which was maintained even after 24 h. As for the hydrophilic intensity and durability, the coating disks were more effective than the uncoated disk, and the thick coating film (TiO2-3) were more effective than the thin coating film (TiO2-1) (Fig. 3). Similar tendencies were observed for the glass disks. The hydrophilic intensity and durability, the coating films were more effective than uncoated disk (Fig. 4). 3.2. Cell attachment and proliferation The observations of cellular proliferation on the TiO2-coated surfaces after 12 h and 3 days of incubation are shown in Fig. 5. At 12 h, initial cellular attachment on the disk surface was seen (Fig. 5A), and cellular proliferation became conspicuous (Fig. 5B, C and D). After 3 days, the cells formed cellular bridges and proliferated (Fig. 5E and I). Sufficient cellular proliferation and cellular bridges were observed on the TiO2coated disks (Fig. 5F, G, H, J, K and L). The numbers of HPLFs on the glass and Ti disk surface after culture for 3 days were shown in Fig. 6 and on the Ti disk at 7 days culture were shown in Fig. 7 respectively. Statistical analysis showed that the number of cells on the TiO2-coated disks was significantly higher than that on the uncoated disks. Further, the number of cells on the thick coating films was significantly higher than that on the thin coating film. The cellular response on the disk surface was dependant on the coated film thickness. 4. Discussion
3.1.4. Contact angle measurement The contact angle measurements for the Ti disks and glass disks were shown in Figs. 3 and 4, respectively. The contact angle of Cont-Ti showed hydrophobicity both with and without
Regarding TiO2 photocatalysts, it is well known that oxidative decomposition promoted by oxidation power of a positive hole generated by photoexcitation [12]. Thereafter,
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Fig. 2. Surface characterisation of the TiO2 coating film. (A) SEM imaging of the surface; scale bar = 100 mm. (B) Surface roughness measurement; the data are the mean SD for all disks (n = 9). Cont-Ti: no coating (control); TiO2-1: 1-mm-thick coating film; TiO2-2: 2-mm-thick coating film; TiO2-3: 3-mm-thick coating film. All surface revealed a similar picture, and no difference was observable among the group. The values were not found to be significantly different between the control and TiO2-coated disks irrespective of the film thickness.
Fig. 3. Time-dependent contact angle of the TiO2-coated surface (Ti disks). The data are the mean SD for all disks (n = 8.). Cont-Ti: no coating (control); TiO2-1: 1-mm-thick coating film; TiO2-2: 2-mm-thick coating film; TiO2-3: 3mm-thick coating film. Shown a picture of the state of the water to drip into the bottom. *All data intervals were significantly different ( p < 0.05). As for the hydrophilic intensity and durability, the TiO2-coated disks were more effective than the uncoated disks, and the thick coating film (TiO2-3) were more effective than the thin coating film (TiO2-1).
Fig. 4. Time-dependent contact angle of the TiO2-coated surface (glass disks). The data are the mean SD for all disks (n = 8). Cont-G: no coating (control); G-TiO2-1: 1-mm-thick coating film; G-TiO2-2: 2-mm-thick coating film; GTiO2-3: 3-mm-thick coating film. Shown a picture of the state of the water to drip into the bottom. *All data intervals were significantly different ( p < 0.05). As for the hydrophilic intensity and durability, the TiO2-coated disks were more effective than the uncoated disks.
Wang et al. [17] found that TiO2 surfaces under UV irradiation produce a hydrophilic surface. After further studies, the mechanism of hydrophilicity development was explained by decomposition and removal of organic compounds by a photogenerated positive hole and hydrophilic domain formation by increase in surface hydroxyl groups [30–32]. At present, TiO2 photocatalysts are used in various industries because of these phenomenal characteristics. To obtain the best characteristics, a TiO2 photocatalyst must be coated on base materials and immobilized without structural change of the base materials. Wet and dry processes can be used to achieve such immobilization. The former requires application of
solvents as a raw material of TiO2 before sintering, whereas the latter can be conducted without application of solvents. In both processes, however, heat application, adhesive layers and special equipments are needed. We used a peroxotitanium solution developed for application to any surface without special equipments and solvents for coating TiO2. This neutral solution can be applied as a translucent coating film without heat application at room temperature [27]. Moreover, as shown in Fig. 2, the coated surface was highly smooth. We confirmed that coating with a thickness at the micrometer level is possible. From these findings, we believe that this technique can provide photo-
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Fig. 5. Representative photographs of HPLF proliferation on the TiO2 coating film (glass disks) at 12 h and 3 days after seeding. Scale bar = 100 mm. Cont-G: no coating (control); G-TiO2-1: 1-mm-thick coating film; G-TiO2-2: 2-mm-thick coating film; G-TiO2-3: 3-mm-thick coating film. At 12 h, initial cellular attachment on the disk surface was seen (A), and cellular proliferation became conspicuous (B–D). After 3 days, the cells formed cellular bridges and proliferation (E and I). Sufficient cellular proliferation and cellular bridges were observed on the TiO2-coated disks (F, G, H, J, K, and L). Arrows indicate the cellular bridges.
Fig. 6. Examination of HPLF proliferation on the TiO2 coating film. (A) TiO2-coated surface at 3 days after seeding (glass disks). (B) TiO2-coated surface at 3 days after seeding (Ti disks). All data are expressed as the mean SD (n = 9 for both types of disks). *All data intervals were significantly different ( p < 0.05). The number of cells on the TiO2-coated disks was higher than that on the uncoated disks. The cellular response on the disk surface was dependant on the coated film thickness.
catalytic effects without affecting the base material surface structure of commercial implants. Regarding the hydrophilic status, as can be seen in Figs. 3 and 4, the same level of slight hydrophilicity was observed at
the different film thicknesses even without UV irradiation. Under UV irradiation, sufficient hydrophilicity was obtained even with thin application of the TiO2 photocatalyst; interestingly, we found that the thick coating films could
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structured water. On that surface, after attachment of plasma glycoproteins, extracellular matrix proteins such as collagen I and fibronectin are attached through various ions. These extracellular matrix proteins are critical for regulating cellular function such as cell adhesion, shape and migration [36,37]. We speculate that after this series of surface layering, fibroblasts finally attached onto the TiO2 surface. Consequently, the thick coating film maintaining hydrophilicity for a long time works effectively on the fibroblasts because of its long-lasting effect on their growth. Therefore, such surface modification with hydrophilicity may promote the formation of implant-circumferential connective tissue rich in fibroblasts and allow acquisition of a wellfunctioning barrier mechanism against the external environment. Application of TiO2 photocatalysts with mucosal peri-implant tissue is of great interest at present and will need further study. 5. Conclusion We consider that the newly developed anatase-TiO2 coating film under UV irradiation markedly improves the conductive response of fibroblasts. Fig. 7. Examination of HPLF proliferation on the TiO2 coating film. TiO2coated surface at 7 days after seeding (Ti disks). All data are expressed as the mean SD (n = 9). *All data intervals were significantly different ( p < 0.05). The number of cells on the TiO2-coated disks was higher than that on the uncoated disks. The cellular response on the disk surface was dependant on the coated film thickness.
retain surface hydrophilicity for a long period. Previous studies have reported that this longevity is established by threedimensional arrangements of the anatase crystal structure with stomata of TiO2 [30,33]. Namely, the hydrophilicity of TiO2 surfaces is by nature relatively low, but with UV irradiation, oxygen deletion-promoting dissociative adsorption occurs on the surface, and then, the surface structure changes with formation of hydrophilic domains accompanied with increase of surface hydroxyl groups. Hence, we suggest that the hydrophilicity following the photocatalytic reaction by UV irradiation is determined by three-dimensional arrangements of the anatase crystal structure included in the coating film and that the thick and high-volume membrane has a larger surface area of hydrophilic domains, resulting in higher hydrophilicity. The existence of a hydrophilic status is advantageous to improve cellular attachment and proliferation. In fact, as can be seen in Figs. 5–7, the HPLFs showed an increasing tendency on the TiO2-coated surface compared with the uncoated disks. Especially, the thick TiO2 film rendered a prominent increasing tendency. The mechanism can be explained as follows. In addition to formation of the hydrophilic domains, obtainment of fresh surfaces due to surface hydrocarbon removal by the photocatalytic reaction of TiO2 [29] and resolution of the organic contents result in higher rates of surface hydroxyl groups on the thick film than the thin film [34,35]. Specifically, because the thick films have high hydrophilicity and surface energy, water is attached first on the surface to form a layer of
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