Storage in aqueous solution or ultraviolet (UV) irradiation can retain or regain the hydrophilicity of titanium implant surface. In this study, 3 types of commercial titanium implants were used: ZBL (ZDI Bone Level), CEL (C-tech Esthetic Line), and modSLA (Straumann SLActive). ZBL and CEL implants were treated with UV irradiation for 4 hours. Surface characterization of the 4 groups (ZBL, ZBL-UV, CEL-UV, and modSLA) was evaluated by scanning electron microscopy and contact angle measurements. The in vivo bone response was evaluated by removal torque (RTQ) tests and histomorphometric analysis at 3, 6, and 12 weeks postimplantation. A total of 144 implants and 36 rabbits were used for experiments according to a previously established randomization sequence. The ZBL-UV, CEL-UV, and modSLA groups were hydrophilic, and nanostructures were observed on the modSLA implant surface. ModSLA achieved better RTQ value than ZBL at 12 weeks (P < .05). For histomorphometric analysis, ZBL-UV and CEL-UV implants showed higher bone area values in the cancellous bone zone at 6 weeks than did modSLA and ZBL implants (P < .05). In the cortical bone zone, all groups showed comparable bone-to-implant contact at all healing time points (P > .05). Both storage in saline and UV irradiation could retain or provoke hydrophilic surfaces and improve osseointegration. Compared with storage in saline, UV irradiation displayed slight advantages in promoting new bone formation in cancellous bone zone at an early stage.
Introduction
Titanium implants have been the gold standard in the field of implantology due to their excellent physical properties, superior biological compatibility, and osseointegration ability.1 Among various implant surface properties, hydrophilicity is a crucial property that influences initial cell behavior and the subsequent osseointegration process.2–5 Freshly manufactured titanium implant surfaces are considered to be superhydrophilic, which means that the contact angle of liquid drops on solids is less than 5°.6,7 However, this characteristic gradually attenuates, with contact angle increasing to greater than 60° in 4 weeks.6,8 After the preparation of titanium implants, the implant surfaces would be exposed in the air and be polluted by hydrocarbons, thus weakening hydrophilicity.6,8
Rupp et al9 has pointed out that storage of implants in an aqueous solution could retain their hydrophilicity and high surface energy. SLActive implant from the Straumann company maintains the treated titanium implants in isotonic sodium chloride (NaCl) solution. In vitro studies have shown that a significant increase of alkaline phosphatase activity and osteocalcin and osteoprotegerin production was found in the SLActive surface.10–13 In addition, SLActive surfaces were suggested to enhance angiogenesis induced by osteoblasts,14 and a significant effect on platelet activation and chemokine release was also found.15 In vivo experiments and clinical studies have also proven that SLActive surface improves bone apposition, osseointegration, and implant stability.16–22
Ultraviolet (UV) irradiation is an alternative way to change material surfaces into their hydrophilic nature.23 Up until recently, there have been 2 explanations regarding this phenomenon. On the one hand, UV irradiation could degrade the absorbed hydrocarbon on implant surfaces effectively.24 On the other hand, oxygen vacancies formed on the surface of titanium dioxide (TiO2) (induced by UV irradiation) could dissociate water in the air and thus produce hydroxyl groups, resulting in hydrophilicity.25 In vitro studies have shown that UV photofunctionalization could increase protein adsorption and cell proliferation, attachment, and migration and promote osteoblastic differentiation and mineralization.26–31 In vivo studies have demonstrated that implants with UV irradiation enhance the bone quality of newly formed bone and promote osseointegration.30,32–34 Clinical studies have also shown that UV photofunctionalization enhances and accelerates implant stability.35–37
Both storage in saline and irradiation by UV have been reported to create hydrophilic surfaces effectively and improve osseointegration. However, there is little published research directly comparing these 2 treatments, and only Ghassemi et al38 have recently reported different biological effects on osteoblastic behavior in vitro. In this study, commercially available titanium implants were used to compare the effects of storage in saline and UV irradiation. Implant surfaces after 2 different treatments were characterized, and their in vivo bone responses were evaluated.
Materials and Methods
Titanium implant preparation
The titanium implants used in this study were commercially provided (Table 1). Both ZDI Bone Level (ZBL) and C-tech Esthetic Line (CEL) were sandblasted, large grit, and acid-etched (SLA) implant surfaces. For further UV treatment, the implants were irradiated by UV using a UV irradiation device (Zhuojing Technology Development Co, Ltd, Tianjin, China) with a wavelength of 250 ± 20 nm. The whole process was performed under ambient conditions at room temperature for 4 hours. SLActive implants (Straumann SLActive [modSLA]) were produced with sandblasting and acid-etching and stored in a sealed glass tube containing isotonic NaCl solution.
Surface Characterization
The surface morphology of titanium implants was analyzed using scanning electron microscopy (SU8010, Hitachi, Tokyo, Japan). One implant of each group was selected randomly and measured at 3 different points. Contact angle analysis was performed with the static sessile drop method using a video-based contact angle system (SL200B, Solon Tech, Shanghai, China). Furthermore, 1 μL of deionized water was dropped on the top of each implant, and the static contact angle was measured, with <5° considered superhydrophilic and >90° considered hydrophobic.7 Since all the specimens had to be desiccated before the experiment, the modSLA group was dried by nitrogen in advance.
Surgical Procedure
Animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Zhejiang University (ZJU20181169), and all procedures were performed according to the Guidelines for Animal Care and Use Standards. A total of 36 adult New Zealand white rabbits (male, weighing 3.0 kg on average, aged 6–8 months) were used for the experiments and were randomly assigned to 3 batches (3, 6, and 12 weeks) based on the identifying number on an ear tab. All of them were housed at room temperature in individual cages and fed a standard rabbit diet. Prior to surgery, a randomization sequence was performed using the calculator (fx-991ES PLUS, Casio Computer Co, Ltd, Shanghai, China), and the rabbits were selected according to this number sequence. These rabbits were anesthetized with intramuscular injections of 3% pentobarbital sodium (30 mg/kg; West Asia Chemical Co, Ltd, Shandong, China). Local anesthesia was induced in the surgical area with 2% lidocaine injection. After successful anesthesia induction, the bilateral tibiae and femur were shaved and disinfected with iodophor and then draped with a sterile fenestrated sheet. Small linear skin incisions were made in the area of the femoral condyles and tibial metaphyses. After blunt dissection of the subcutaneous tissue, the surgical surfaces were exposed. A sequence of rotary drills was used to create a hole in the femoral condyles and tibial metaphyses according to the protocol provided by manufacturers. The drilling sequence of ZBL was drill D2.2 (800 rpm), D2.7 (600 rpm), and D3.4 (400 rpm). The drilling sequence of CEL was drill D2.1 (1000 rpm), D3.1 (800 rpm), and D3.5 (600 rpm). The drilling sequence of modSLA was drill D2.2 (800 rpm), D2.8 (600 rpm), and D3.5 (500 rpm). Subsequently, holes were enlarged to 3.4 mm (ZBL, ZBL-UV), 3.5 mm (CEL-UV), and 3.5 mm (modSLA) in diameter with 0.9% NaCl solution irrigation. The implants were gently screwed into corresponding holes at 15 rpm, and the shoulder of the implants was at the same level as the cortical bone (Figure 1a and b). Overall, 144 implants were implanted in the femoral condyles and tibiae of 36 rabbits. The surgical area was stratified sutured, and the animals received antibiotics intramuscularly (penicillin, 400,000 U/d) (Harbin Pharmaceutical Group, Heilongjiang, China) for 3 days.
Surgical procedures: (a) incisions in the femoral condyle (upper) and tibial metaphyses (lower) and (b) implant and healing abutment after implantation.
Surgical procedures: (a) incisions in the femoral condyle (upper) and tibial metaphyses (lower) and (b) implant and healing abutment after implantation.
The rabbits were sacrificed using anesthetic overdose at 3, 6, and 12 weeks after surgery. For removal torque (RTQ) tests, the implants inserted in the femoral condyles were used. For histomorphometric analysis, the implants in the tibia were used. The data were blindly collected and analyzed to reduce researcher bias.
RTQ Measurements
After 3, 6, and 12 weeks of healing, the femoral condyles with implants were harvested and fixed to an electronic torsion testing machine with 47°C melting point indium-tin alloy, similarly to a previous study (CTT2500, MTS Company, Eden Prairie, MN).39 The speed of the machine was controlled at 5°/min. The maximum binding strength was determined by the peak value of the RTQ curve.
Histological Examination
The tibial metaphyses of rabbits with implants were dissected and immersed in 4% paraformaldehyde (Solarbio Technology Co, Ltd, Beijing, China) for 7 days. Sections were cut to 200 μm thickness and polished to 20–30 μm. Subsequently, these samples were stained in Stevenel blue and van Gieson picro-fuchsin. A bright-field microscope DM4000 (Leica, Wetzlar, Germany) and image analysis system (Image Pro Plus, Media Cybernetics, Rockville, MD) were used in histological examination. Bone-to-implant contact (BIC) and the percentage of bone area (BA) in the cancellous and cortical bone were measured. Bone-to-implant contact was calculated by dividing the implant length by the length of the bone in direct contact with the implant surface. Bone area was measured as the percentage of bone located within 200 μm of the implant surface, according to a previous study.17
Statistical Analyses
Mean values and SDs were used to calculate the parameter. One-way analysis of variance was performed to examine the data from different groups at same time points, P < .05 was considered statistically significant, the equality of variances was verified using Levene test, and Bonferroni correction was used as the post hoc test. The SPSS 20.0 software (Chicago, IL) was used for all statistical analyses. The results were reviewed by an independent statistician (Wen Yuanyuan, associate chief physician, Hangzhou Center for Disease Control and Prevention).
Results
Surface characterization
The surface topography of the titanium implant was similar among the ZBL (Figure 2a–c), ZBL-UV (Figure 2d–f), and CEL-UV groups (Figure 2g–i), presenting a microtopographic configuration with irregular peaks and pits (0.5–1.5 μm). Contrarily, scattered particles with a diameter of 50 nm were observed on the surface of the modSLA group (Figure 2j–l).
Scanning electron microscopy images of ZBL (a, b, c), ZBL-UV (d, e, f), CEL-UV (g, h, i), and modSLA (j, k, l) at 5000 magnification (a, d, g, j), 20,000 magnification (b, e, h, k), and 100,000 magnification (c, f, i, l). CEL indicates C-tech Esthetic Line; modSLA, Straumann SLActive; UV, ultraviolet; ZBL, ZDI Bone Level.
Scanning electron microscopy images of ZBL (a, b, c), ZBL-UV (d, e, f), CEL-UV (g, h, i), and modSLA (j, k, l) at 5000 magnification (a, d, g, j), 20,000 magnification (b, e, h, k), and 100,000 magnification (c, f, i, l). CEL indicates C-tech Esthetic Line; modSLA, Straumann SLActive; UV, ultraviolet; ZBL, ZDI Bone Level.
Contact Angle
The contact angle represents the hydrophilicity of an implant surface. The implants from the ZBL-UV, CEL-UV, and modSLA groups showed a contact angle of almost 0°, indicating superhydrophilic surfaces, whereas ZBL implants were hydrophobic (>100°) (Figure 3).
Implant hydrophilicity of ZBL-UV (a, b), CEL-UV (c, d), modSLA (e, f), and ZBL (g, h) before and after examination. CEL indicates C-tech Esthetic Line; modSLA, Straumann SLActive; UV, ultraviolet; ZBL, ZDI Bone Level.
Implant hydrophilicity of ZBL-UV (a, b), CEL-UV (c, d), modSLA (e, f), and ZBL (g, h) before and after examination. CEL indicates C-tech Esthetic Line; modSLA, Straumann SLActive; UV, ultraviolet; ZBL, ZDI Bone Level.
RTQ Measurements
Removal torque measurement results represented the stability of the implant-bone bond. The torque value gradually increased with time. The torque values of the modSLA group were the highest at 3, 6, and 12 weeks, whereas the ZBL group had the lowest torque values. However, there were no significant differences in RTQ values among the groups examined, except at 12 weeks between the modSLA and ZBL groups (P < .05) (Table 2).
Histological and Histomorphometric Analysis
Microscopic images of implants with the bone in the cancellous zone are shown in Figure 4. The bone was in direct contact with the implant surface in 3 weeks in all the groups, and no evident inflammation was observed. As time progressed, the bone volume increased significantly in all groups. New bone adhered to the surface of the implant and gradually changed from reticular and strip bone in 3 weeks to lamellar bone in 12 weeks. The amount of bone trabecula and bone matrix was lower in the ZBL group than in the other groups regardless of healing time. The histological results of the ZBL-UV, CEL-UV, and modSLA groups were similar at 3, 6, and 12 weeks. Magnified microscopic images of implants with the bone in the cortical zone are shown in Figure 5. The cavity between the cortical bone and the implant gradually reduced, and the newly formed bone became denser, displaying the morphology of the lamellar bone. No evident differences were observed among the 4 groups.
Light micrographs of ZBL-UV (a, b, c), CEL-UV (d, e, f), modSLA (g, h, i), and ZBL (j, k, l) at 3 (a, d, g, j), 6 (b, e, h, k), and 12 (c, f, i, l) weeks in the cancellous bone. CEL indicates C-tech Esthetic Line; modSLA, Straumann SLActive; UV, ultraviolet; ZBL, ZDI Bone Level.
Light micrographs of ZBL-UV (a, b, c), CEL-UV (d, e, f), modSLA (g, h, i), and ZBL (j, k, l) at 3 (a, d, g, j), 6 (b, e, h, k), and 12 (c, f, i, l) weeks in the cancellous bone. CEL indicates C-tech Esthetic Line; modSLA, Straumann SLActive; UV, ultraviolet; ZBL, ZDI Bone Level.
Light micrographs of ZBL-UV (a, b, c), CEL-UV (d, e, f), modSLA (g, h, i), and ZBL (j, k, l) at 3 (a, d, g, j), 6 (b, e, h, k), and 12 (c, f, i, l) weeks in the cortical bone. CEL indicates C-tech Esthetic Line; modSLA, Straumann SLActive; UV, ultraviolet; ZBL, ZDI Bone Level.
Light micrographs of ZBL-UV (a, b, c), CEL-UV (d, e, f), modSLA (g, h, i), and ZBL (j, k, l) at 3 (a, d, g, j), 6 (b, e, h, k), and 12 (c, f, i, l) weeks in the cortical bone. CEL indicates C-tech Esthetic Line; modSLA, Straumann SLActive; UV, ultraviolet; ZBL, ZDI Bone Level.
The BA value in the cortical bone showed no significant difference among the ZBL-UV, CEL-UV, and modSLA groups, at 3, 6, or 12 weeks, but the ZBL-UV and modSLA groups showed higher BA values in the cortical bone than did the ZBL group at 12 weeks (P < .05) (Figure 6). ZBL-UV implants showed higher BA value than modSLA implants at 3 weeks in the cancellous bone (18.01 ± 1.22 vs 13.72 ± 0.68, P < .01). ZBL-UV and CEL-UV implants showed higher BA values in the cancellous bone than modSLA implants at 6 weeks (22.90 ± 2.17, 20.98 ± 1.36, and 18.05 ± 1.28, respectively; P < .05). Further, at 12 weeks, there was no significant difference among the 3 groups (25.44 ± 3.21, 25.96 ± 2.11, and 28.08 ± 2.42, respectively; P > .05), and the ZBL-UV, CEL-UV, and modSLA groups showed higher BA values in the cancellous bone than the ZBL group (P < .01) (Figure 7).
Bone area in the cortical bone within 200 μm around the implant in the 4 groups at 3, 6, and 12 weeks. n = 6; *P < .05, **P < .01.
Bone area in the cortical bone within 200 μm around the implant in the 4 groups at 3, 6, and 12 weeks. n = 6; *P < .05, **P < .01.
Bone area in the cancellous bone within 200 μm around the implant in the 4 groups at 3, 6, and 12 weeks. n = 6; *P < .05, **P < .01.
Bone area in the cancellous bone within 200 μm around the implant in the 4 groups at 3, 6, and 12 weeks. n = 6; *P < .05, **P < .01.
There was no significant difference among groups in BIC regardless of the healing time. However, the mean value in the ZBL group was the lowest among the 4 groups. Figures 8 and 9 show the representative images of BIC in the cortical and cancellous bones at 3, 6, and 12 weeks.
Bone-to-implant contact in the cortical bone section in the 4 groups at 3, 6, and 12 weeks. n = 6.
Bone-to-implant contact in the cortical bone section in the 4 groups at 3, 6, and 12 weeks. n = 6.
Bone-to-implant contact in the cancellous bone section in the 4 groups at 3, 6, and 12 weeks. n = 6.
Bone-to-implant contact in the cancellous bone section in the 4 groups at 3, 6, and 12 weeks. n = 6.
Discussion
In this study, the contact angle demonstrated that both UV irradiation and storage in saline could effectively modify implant surfaces into being hydrophilic, which is consistent with the results of other studies.6,9,40 Bone histomorphometric analysis and RTQ test were performed to further evaluate the in vivo response. Compared with the ZBL group, the ZBL-UV, CEL-UV, and modSLA groups displayed significantly higher BA in the cancellous bone. Regarding BIC, the ZBL group also showed the lowest mean value among the 4 groups in the cortical and cancellous bones regardless of the healing time. The RTQ results also demonstrated the lowest value in the ZBL group. These results demonstrated that both UV irradiation and storage in saline were able to improve osseointegration, a result consistent with that of other studies.16,17,26,34
The histomorphometric results displayed a significant difference between groups in the cancellous bone, but similar results in the cortical bone. This may be due to the differences in bone remodeling, glycation, and pharmacokinetics between the cortical and cancellous bone region.41–43 A previous study has also shown similar percentage contact of the cortical bone and bone volume of different implant surfaces.44 Implant surface had a greater effect on the cancellous bone than the cortical bone,45–47 which could possibly be the reason why no significant difference was found among implant surfaces in the cortical region.
For comparison between the UV irradiation group and the saline storage group, ZBL-UV and CEL-UV showed significantly higher BA in the cancellous bone in the early healing time at 3 and 6 weeks, indicating slight advantage over saline storage in terms of early osseointegration. An in vitro study by Ghassemi et al38 has displayed higher levels of osteogenic gene expression and mineralization of osteoblasts cultured on UV-treated titanium compared with saline-stored samples.
The difference between the UV-treated groups and the modSLA group during the early healing time may not be caused by hydrophilicity alternation but by other alternations on implant surfaces. The reasons may be as follows: First, hydroxyl radicals formed after UV irradiation of TiO2 had strong oxidizing property, which was sufficient to degrade the contaminated hydrocarbons on the implant surface.48 Compared with saline storage, UV irradiation showed less carbon on the implant surface.38 Isolation of hydrocarbon contamination enabled direct attachment of proteins and cells to the implant surface, which facilitated the healing process.49 Second, the terminal hydroxyl group, which increased after UV irradiation, could enhance protein adsorption by electric attraction with the amino group in proteins, leading to the improvement of subsequent osteoblast adhesion and mineralization.31 Third, the exposure of TiO2 after UV treatment made the implant surface electropositive through excitation of an electron from the valence band to the conduction band. In this way, the implant surface changed from cathode to anode and allowed direct contact between proteins and TiO2 without divalent cation mediation.28 Compared with the saline-stored surface, more cells attached on the UV irradiation surface, resulting in more calcium depositions.38 A previous study has also suggested that the effect of hydrophilicity may be overwhelmed by the influence from isoelectric control or anion treatment.28
The highest mean torque was found in the modSLA group. The reason may be related to the nanoparticle structures formed on the modSLA implant surface.50 A recent study has indicated that implant nanotopography has a greater contribution to the overall bone anchorage than hydrophilicity.51 The effects of nanostructures on osteoblast behavior and bone formation have been reviewed.52 Micro-/nanostructure topographies have been reported to improve cell adhesion and osteogenic differentiation in vitro53,54 and improve osteogenesis ability in vivo.55 In the present study, the result of torque removal was consistent with the histological result of BIC and BA in the cortical bone, which was consistent with results by van Oirschot et al.56 Cortical tissues were dense, whereas the cancellous bone was relatively loose, which leads to differences in mechanical properties.57 It is the cortical bone, not the cancellous bone, that determines the peak torque value.58
The present study has some limitations. With the anatomic, metabolic, and cellular differences between animals and humans, the animal models do not reliably predict results in humans, and implants placed in long bones might not behave similarly to implants placed in jawbones.59,60 In addition, the wavelength and irradiation time of the UV irradiation device were different from those in previous studies that used TheraBeam Super Osseo (Ushio, Tokyo, Japan) for 15 minutes32,34–36 or 15-W bacterial lamp for 48 hours.26–28 Further studies on the optimal parameters such as intensity and time for UV irradiation are needed.
In summary, the results of the present study revealed that hydrophilicity created by UV irradiation could improve osseointegration at an early stage, which suggests that the use of photofunctionalization may allow for a faster loading protocol. Moreover, UV irradiation is effective on all surface topographies of Ti-based materials, implying versatile applicability to a wide range of dental implants.26,27 Considering the simple operation and low cost of the method, chairside UV irradiation may be used to promote osseointegration before implant placement.
Conclusion
Both saline storage and UV irradiation-induced hydrophilic surfaces could achieve sound in vivo bone response in a rabbit model compared with an SLA surface. However, a UV irradiation surface may improve early bone formation in cancellous BAs compared with saline storage surface.
Abbreviations
Acknowledgments
Implants were provided by Institut Straumann AG, Basel, Switzerland, C-tech Implant, Srl., Bologna, Italy and Zhejiang Guangci Medical Appliance Co., Ltd., Ningbo, China. This research was funded by National Natural Science Foundation of China (No.31670970), Science and Technology Department of Zhejiang Province (No. 2019C03081). The authors also thank Hao Bai for performing the statistical analysis.
References
Note The authors declare no conflicts of interest.