The aim was to investigate the osseointegration of a novel coating—plasma-sprayed nanostructured zirconia (NSZ)—for dental implants. Nanostructured zirconia coating on non-thread titanium implant was prepared by plasma spraying, and the implant surface morphology, surface roughness, and wettability were measured. In vivo, nanostructured zirconia-coated implants were inserted in rabbit tibia, and the animals were sacrificed at 2, 4, 8, and 12 weeks after implantation. The bond strength between implant and bone was measured with the removal torque (RTQ) test. Osseointegration was observed by scanning electron microscopy (SEM), microcomputerized tomography (micro CT), and histological analyses. Quantified parameters were calculated, including removal torque, bone volume to tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation/spacing (Tb.Sp), and bone-implant contact (BIC) percentage. The statistical differences were detected with a two-tail Mann-Whitney U test (SPSS 20.0). The surface roughness (1.58 μm) and wettability (54.61°) of a nanostructured zirconia-coated implant was more suitable than the titanium implant (0.598 μm, 74.38°) for osseointegration and hierarchical surface morphology seen on the zirconia coating. The histological analyses showed that a zirconia-coated implant induced earlier and had more condensed bone formation than did the titanium implant at 2 and 4 weeks. Quantified parameters showed the significant differences between these 2 groups at an early healing period, but the differences between the 2 groups decreased with an increased healing period. All these results demonstrated that plasma-sprayed zirconia coated implants induced better bone formation than did titanium implants at an early stage.
Titanium (Ti) and titanium alloy implants are currently used in oral implantology for restoration in edentulous patients. They have achieved success due to their excellent biocompatibility, great mechanical properties, corrosion resistance, and rapid osseointegration.1–3 However, there are some reports that Ti causes allergic reactions in the human body4 and that a Ti implant may generate galvanic effects after contacting saliva.5 In addition, Ti wear particles can also lead to tumor necrosis factor alpha-mediated inflammatory responses.6 It has been confirmed that aluminum (Al) and vanadium (V) ions will be released from Ti-6Al-4V, especially in inflammatory conditions.7,8 Moreover, compromised esthetics is another problem in need of a solve for Ti and Ti alloy implants.9 To overcome the intrinsic disadvantages of Ti dental implants, high strength, and biocompatibility ceramics (such as zirconia) were employed to fabricate implants.
Zirconia possesses high bending strength (900–1200 MPa), excellent hardness (1200 Vickers), low heat conductivity, great corrosion resistance, and biocompatibility, decreasing the aggregation of platelets. Among all kinds of zirconia, yttria-stabilized tetragonal zirconia polycrystals (Y-TZP) is the main component of zirconia implants and could be used as coating material for Ti implants because of its stress-induced phase transformation toughing theory, improving the fracture resistance of zirconia coating.10–12 Zirconia has been used in dental implantology as an implant material and presented similar, even superior, osseointegration ability to that of Ti implants13–17 with biosecurity and esthetics of implants improved at the same time. Vulnerability to fracture was observed in zirconia implants because of the high brittle property of zirconia;18 thereafter, zirconia was used as coating material for implants to avoid fracture, and many surface modifications were used to improve the chemical composition or the topography of zirconia's surface when interacting with soft and hard tissues. The mainstream surface modification strategies can be generalized into three categories: physical (blasting, plasma spraying, ion implantation, laser treatment, pulsed magnetron sputtering), chemical (acid etching, anodic oxidation, microarc oxidation), and biological (protein absorption, ionic interaction).19–21
To date, sandblasting or sandblasting with acid etching (SLA), dip coating, and plasma spraying have been used as the main methods of surface modification on Ti implants with zirconia.22–26 Plasma spraying is powerful tool for surface modification, with a wide range of options in coating materials, such as metal, ceramic, and composites. Plasma-sprayed coatings can significantly improve the physical and chemical properties of substrate materials, and the surface morphology, roughness, porosity, elemental composition, and degree of crystallization can be easily controlled by manipulating relevant parameters.
In our previous study, we employed a plasma-spraying technique and nanostructured zirconia (NSZ), preparing a zirconia coating on a Ti abutment, and obtaining satisfactory outcomes in mechanical property and compatibility. In this study, we investigated the application of plasma-sprayed NSZ coating in implants.
Materials and Methods
Surface analyses of NSZ-coated dental implants
Non-threaded dental implants (length 5 mm, inner diameter 1.5 mm, outer diameter 2.5 mm, Norman Metal Products, Qingdao, China) were produced from medical grade Ti bars (Baoji Titanium Industry Co, Shaanxi, China; Figure 1a). All Ti implants were polished and rinsed with tap water for 30 minutes. Yttria-stabilized tetragonal zirconia pelleting powder (Y-TZP) with the particle size ranging from 70 to 110 nm (Lida Hi-Tech Special Material Co, Ltd, Changshu, Jiangsu, China) was heat-treated and spray-dried to form circular or oval-shaped particles (15∼45 μm). The pelleted NSZ particles were then plasma sprayed under low vacuum onto the surface of Ti implants (SM-80 Plasma spraying system, Xiuma Spraying Machinery Co, Ltd, Shanghai, China; Figure 1b). The main parameters of plasma spraying are as follows: arc voltage 82V, arc current 500A, air flow rate 2.1 m3/h, powder feed rate 40 g/min, and spray distance 70 mm.
The NSZ-coated implants were used for surface analyses and the animal study. The surface roughness of NSZ-coated implants was measured by portable Roughometer (MarSurf PS1, Elcometer Ltd, Manchester, UK), and the surface microstructure of NSZ coating layer was analyzed by SEM (JSM-6380 LA). A Drop Shape Analyzer (DSA100, KRÜSS, Hamburg, Germany) was used to evaluate the wetting of NSZ coating surface by artificial saliva, which was quantified by averaging the contact angles on two opposite sides of the saliva drop.
Measurements of the surface roughness and artificial saliva contact angle were conducted 3 times on 6 NSZ-coated implants. The statistical differences between Ti implants and NSZ-coated implants in surface roughness and contact angle were detected by Student t test (SPSS 20.0, Chicago, Ill), and the differences in removal torque (RTQ), bone-implant contact (BIC), bone volume to tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation/spacing (Tb.Sp0 were detected by a two-tail Mann-Whitney U test (SPSS 20.0). The significance level was 0.05.
Assessment of osseointegration induced by NSZ-coated dental implants
The Ethics Committee of Shandong University approved this study protocol (NO. GD201615). Sixty-eight New Zealand white rabbits (male, 2.5∼3.0 kg) were randomly divided into 4 groups (A–D, n = 17). After general anesthesia with 10% chloral hydrate (1.5 ml/kg), each animal received implant surgery on its tibia close to the knee joint, with a Ti implant placed on the right side and a NSZ-coated implant on the left. To facilitate the insertion of implant into the bone, a pilot hole was prepared with Marathon Implant Motor (Ki-20, Saeyang, Daegu, Korea). Intramuscular injection of cefazolin was conducted for 3 consecutive days after surgery. The 4 groups of animals were sacrificed at 2 weeks (A), 4 weeks (B), 8 weeks (C), and 12 weeks (D), and implants with about 5 mm surrounding bone were dissected with an electric bone drill (Small Bone Tools, BioAccess, Baltimore, Md).
From each group of animals, 8 pairs (right and left side) of freshly dissected bone samples were used for RTQ analysis on Electromechanical Universal Tester (Shimadzu Scientific Instruments, Columbia, Md) within 24 hours. Three pairs of samples were sectioned along the long axis and through the center of the implants. The exposed surfaces were analyzed by scanning electron microscope (SEM) scanning (JSM-6380 LA). The remaining bone samples from the other 6 animals were embedded in light-cure resin (Technovit 7200VLC, Hereaus Kulzer, Wehrheim, Germany) after being fixed in 4% PFA for a week, and 30-μm sections vertical to the long axis of implant were prepared. Tissue morphology around the implant was analyzed under light microscope following methylene blue-acid fuchsin staining. The protocol of histological analysis was similar to that described previously.27–29 Percentage of BIC was calculated using Image-Pro Plus (Media Cybernetics, Rockville, Md) based on the histology images.
Micro CT scanning was carried out (Inveon Multi-Modality System, 80kV, 500 μA, 360° rotation, 18 μm resolution, Siemens, Munich, Germany) for the tissue blocks before they were sectioned for histological analysis. Dicom files were imported into Inveon Research Workplace (Siemens) for 3D reconstruction. Four parameters—including (%), Tb.Th (mm), Tb.N (1/mm) and Tb.Sp (mm)—were generated by the software for regions of interest that incorporated the tissues 1 mm apart from the bone-implant interface.
The measurements of RTQ, BIC, BV/TV, Tb.Th, Tb.N, and Tb.Sp for NSZ-coated implants (left side) were compared with those obtained for implants without surface modification (right side). The comparisons between two groups (left and right sides) were conducted separately for the 4 different healing stages: 2 weeks, 4 weeks, 8 weeks, and 12 weeks. The statistical differences were detected by two-tail Mann-Whitney U test (SPSS 20.0) with a significance level of .05.
The non-threaded Ti implants used for surface property analyses and osseointegration assessment were coated with 400 μm NSZ. In the first part of the surface analyses, the average surface roughness of NSZ coating layer was 1.58 ± 0.21 μm (Table 1), which was significantly higher than that of Ti implants (0.598 ± 0.11 μm, t test, n = 18, t = 12.438, df = 35, P < .05; Figure 2a). Under SEM, the surface of the NSZ coating layer showed a hierarchical structure characterized by microporous structures with the diameters ranging from 5 μm to 10 μm and nanoporous structures with the diameter about 1 μm; the micro-/nanopores were distributed in a regular pattern (Figures 2c3 and c4). By comparison, the microstructures on the Ti surface were more evenly scattered with a lack of pores (Figures 2c1 and c2). In addition, the contact angle of the NSZ coating surface was significantly lower than that of the mechanically processed Ti surface (NSZ: 54.61 ± 1.80°, Ti: 74.38 ± 3.61°, t test, t = 37.198, df = 35, P < .05; Figure 2b).
In vivo osseointegration assessment
For osseointegration assessment, RTQ values (N/cm) were first recorded for NSZ-coated (NSZ+Ti) and Ti implants placed in rabbit tibia at 4 stages of the healing period: 2 weeks, 4 weeks, 8 weeks, and 12 weeks after implant surgery. From 2 weeks to 8 weeks, the RTQ values increased steadily in both groups, peaking at 8∼12 weeks (Figure 3a). Statistically significant differences were detected between NSZ+Ti and Ti groups at 4 weeks, 8 weeks, and 12 weeks (Mann-Whitney U test, n = 8, Mann-Whitney U = 0, Z = −3.361, P < .05; Figure 3a). To be specific, the RTQ values in NSZ+Ti group were significantly higher than those in Ti group.
SEM, microcomputerized tomography (micro CT), and histological analyses showed similar results (Figure 3b, c, and h). Histological analyses revealed that in NSZ+Ti groups, bone/bone-like structures were already deposited along the surface of implants with few gaps between bone and implant at 2 weeks. Compared with NSZ+Ti implants, the arrangement of bone trabeculae in Ti implants were sparse, with more gaps and fibrous tissue seen. By the end of 4 weeks, the architecture of newly formed bone became more condensed and regular in NSZ+Ti group than in the Ti group. At 8 weeks, the gaps and fibrous tissue around implants reduced, and high-organized lamellar bone could be seen; however, in some areas of Ti implant, new bone was still sparse. After the 12 weeks of healing period, close contact formed in NSZ+Ti implant with some gaps still seen between the Ti implant and bone (Figure 3h). The quantified parameters of NSZ+Ti groups, including BV/TV (%) (Mann-Whitney U test, n = 6, Mann-Whitney U = 0, Z = −2.882, P < .05), Tb. Th (mm) (Mann-Whitney U test, n = 6, Mann-Whitney U = 0, Z = −2.882, P < .05), Tb. N (1/mm) (Mann-Whitney U test, n = 6, Mann-Whitney U = 0, Z = −2.882, P < .05), and BIC (%) (Mann-Whitney U test, n = 6, Mann-Whitney U = 0, Z = −2.882, P < .05), were significantly higher than Ti group at 2 weeks, 4 weeks, 8 weeks, and 12 weeks of the healing period (Figures 3d through f, and i). Significant differences in Tb. Sp (mm) (Mann-Whitney U test, n = 6, Mann-Whitney U = 0, Z = −2.882, P < .05) between the two groups were only detected at 2 weeks, 4 weeks, and 8 weeks (Figure 3g). The descriptive statistics of RTQ, BIC, BV/TV, Tb.Th, Tb.N, and Tb.Sp of NSZ-coated and Ti implants are shown in Table 2.
In this study, the osseointegration of plasma-sprayed NSZ-coated implant was shown to be superior to titanium implants in all the quantified parameters, and enhanced osseointegration was induced by NSZ-coated implants more than in titanium implants.
Because of the excellent mechanical properties and biocompatibility of plasma-sprayed NSZ coating in our previous study, plasma spraying was employed to prepare NSZ coating on polished pure titanium implants. The contact angle of artificial saliva on NSZ and titanium implants were 54.61° and 74.38°, respectively. The lower contact angle means better wettability and higher surface free energy for the NSZ-coated implant. Proteins, such as fibronectin, tended to attach on the surface with high free energy, and more arginine-glycine-aspartate triple peptide of fibronectin (as the ligand combined with the integrin of osteoblasts), leading to more attachment of osteoblasts on the NSZ-coated implant at an early stage.30,31 At 2 weeks, it could be seen from histological analysis that more osteoblasts attached on the NSZ-coated implant and more new bone formed around the NSZ-coated implant in the Micro CT image. The same situation could be found in BIC at 2 weeks. These findings were consistent with the theory that a surface with higher free energy promoted the attachment of osteoblasts.32
Moreover, the surface roughness of NSZ coating was 1.58 ± 0.21 μm, much higher than 0.598 ± 0.11 μm of a polished pure titanium implant. Evidence from in vivo and in vitro studies found that a rough surface had a positive effect on osseointegration, including osteoblast attachment, proliferation, and differentiation.33,34 Zhuang and Galli found that compared with smooth surface, SLA surface regulated the activation of osteogenic relative signal pathway, such as ERK1/2 and Wnt/β-catenin signal pathways, promoting the osteogenic differentiation of mesenchymal cells.35,36 In addition, some studies found that when the surface became smooth, osteoblastic cells showed a significant decrease of alkaline phosphatase activity.37,38 These findings all suggested that a rougher surface possessed better ability to induce osseointegration than did a smooth surface. It could be seen in SEM that NSZ-coated implants showed better osseointegration than titanium implants; at 8 weeks, there was a blurry transition layer between the NSZ coating and bone, but there was still a distinct boundary in the titanium implant.
Another notable finding was that plasma-sprayed NSZ coating presented a hierarchical surface, micro-scale surface roughness and a nanoscale porous surface, which was confirmed to be good for attachment, proliferation, and differentiation of osteoblasts. Gittens et al found that nanostructures alone might regulate the proliferation of osteoblasts, but differentiation of osteoblasts would not be affect by nanostructures without microscale structure; the combination of nano-/microscale structure produced a synergistic effect on cell proliferation and differentiation.39 Huang et al studied the behavior of osteoblast-like cells on nano- and microscale structured surfaces, concluding that a nanoscale structure was beneficial to the anchoring of filopodia to migration.40 In addition, bone marrow mesenchymal stem cells (BMSCs) protruded its filopodia randomly on a smooth surface, without lamellipodia formation which led to the migration of cells. While on a patterned surface, plenty of filopodia of BMSCs anchored, and lamellipodia soon formed.41 Moreover, some theories stated that the hybrid nano-/microscale surface morphology of an implant could promote the osteogenic differentiation of mesenchymal cells.42,43 In this current study, NSZ-coated implants possessed higher values in BIC over the whole healing period compared to the titanium implants, perhaps because the NSZ-coated implant surface promoted anchoring of osteoblast filopodia and formation of lamellipodia, which led to the migration and mineralization of osteoblasts to cover more area of the NSZ-coated implant.
In RTQ, Ti implants and NSZ-coated implants showed similar value at 2 weeks, but the value of NSZ-coated implants increased sharply, with a significant difference appearing between the NSZ-coated implant and the Ti implant at 4 weeks. Relative changes appeared in Tb.N and Tb.Sp at 4 weeks, with a distinct increase and decrease, respectively. The trabecula number and trabecula space of the NSZ-coated implant rapidly came to a high level at 4 weeks, but that of titanium implant progressed slowly, indicating more new bone and earlier osseointegration forming around the NSZ-coated implant in the early healing period. From 4 weeks on, the difference between the NSZ-coated implant and the Ti implant decreased with the increase in healing period; at 12 weeks, the NSZ-coated implant and the Ti implant showed similar bone trabecula thickness and bone trabecula space, indicating a similar bone quality and bone trabecula arrangement. This phenomenon indicated that the performance of the NSZ-coated implant and the Ti implant in osseointegration would arrive at the same level later in the healing period. The same progress could be found in previous studies, with the zirconia implant showing similar osseointegration to that of the titanium implant, even though the zirconia implant presented better and earlier osseointegration at an early stage; in the latter healing period, these two kinds of implants showed similar osseointegration in BIC or RTQ.44–46 It seems that different surface modifications and coatings were imperative for early osseointegration but had less effect on the ultimate osseointegration ability. Even so, a better primary stability for the implants was still worthy of study, for better osseointegration and a higher success rate.47,48
In this study, we prepared an NSZ coating that possessed suitable surface parameters and morphology for implantation in a rapid and convenient way (plasma spraying) and without other surface modification, avoiding damage to the integrity and strength of the NSZ coating and presenting excellent osseointegration ability, especially in the early healing period, indicating that plasma-sprayed NSZ coating is a potential candidate for further clinical application. In further study, we will focus on the optimization of plasma-spraying parameters to find the most suitable surface morphology for osseointegration.
An NSZ coating was prepared on polished pure titanium implant by plasma spraying to present a more suitable surface roughness (1.58 ± 0.21 μm) and better wettability compared to the polished titanium implant, Further, a nano-/microscale surface morphology formed on the NSZ coating. In vivo, plasma-sprayed NSZ coated titanium implant induced earlier peri-implant osseointegration than did the titanium implant.
bone marrow mesenchymal stem cell
bone volume to tissue volume
- micro CT
scanning electron microscopy
sandblasting with acid etching
yttria-stabilized tetragonal zirconia polycrystals
The work was funded by grants 81671025 (Beijing, China) from the National Natural Science Foundation of China, 2015GSF118186 (Jinan, China) from the Foundation of Department of Science and Technology of Shandong Province, and the Science and Technology Development Project of Shandong Province 2014GGH218035 (Jinan, China). We thank Dr Guiyong Xiao (School of Material Science and Engineering, Shandong University, China) and Dr Xuetao Yue (Shandong Jianzhu University, China) for their assistance in mechanical tests, implant design, and processing. We also thank Dr Shu Li (Department of Periodontics, Shandong University/Shandong Provincial Key Laboratory of Oral Biomedicine, China) for his facilitation in biosafety training and equipment management.
All authors declare no conflicts of interest.
These authors are co-first authors.