Bone responses to zirconia implants with a thin carbonate-containing hydroxyapatite coating using a molecular precursor method Masatsugu Hirota,1 Tohru Hayakawa,2 Chikahiro Ohkubo,1 Mitsunobu Sato,3 Hiroki Hara,3 Takeshi Toyama,4 Yasuhiro Tanaka5 1
Department of Removable Prosthodontics, Tsurumi University School of Dental Medicine, Kanagawa, Japan Department of Dental Engineering, Tsurumi University School of Dental Medicine, Kanagawa, Japan 3 Division of Liberal Arts, Kogakuin University, Tokyo, Japan 4 Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, Tokyo, Japan 5 Engineering Materials Science, Department of Advance Materials Science, Faculty of Engineering, Kagawa University, Kagawa, Japan 2
Received 17 September 2013; revised 16 December 2013; accepted 7 January 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33112 Abstract: Thin carbonate-containing hydroxyapatite (CA) films coating partially stabilized zirconia (Y-TZP) were prepared (CA-Y-TZP) to establish a metal-free implant system. CA was coated using a molecular precursor method. The CA film was deposited on the surface of Y-TZP using a precursor solution, which was a mixture of a calcium–ethylenediaminetetraacetic acid (EDTA) complex and phosphate compounds. The deposited CA film was characterized by X-ray diffraction, Fourier transform infrared spectroscopy, and energy dispersive X-ray spectroscopy measurements. A focus ion beam system technique revealed that the thickness of the CA film was less than 1.0 mm. Biological evaluations of CA-Y-TZP were performed by immersion experiments in simulated body fluid (SBF) and implantation experiments in the tibiae
and femoral condyles of rabbits. In the SBF immersion experiment, apatite deposition progressed more on CA-Y-TZP at the early stage of immersion than on Y-TZP without the CA coating. Animal experiments revealed that bone formation on CA-Y-TZP was similar with than on Y-TZP. Histomorphometrical evaluations showed a significantly higher boneto-implant contact ratio and bone mass on CA-Y-TZP after implantation into the femoral trabecular bone of rabbits. Therefore, CA-Y-TZP appears to be applicable as a metal-free C 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: implant. V Appl Biomater 00B:000–000, 2014.
Key Words: dental implants, partially stabilized zirconia, apatite coating, simulated body fluid, bone, implant contact
How to cite this article: Hirota M, Hayakawa T, Ohkubo C, Sato M, Hara H, Toyama T, Tanaka Y. 2014. Bone responses to zirconia implants with a thin carbonate-containing hydroxyapatite coating using a molecular precursor method. J Biomed Mater Res Part B 2014:00B:000–000.
INTRODUCTION
Dental implants have been extensively used for fixed, removable, and maxillofacial prosthodontic rehabilitation with a sufficient success rate. The first choice of implant materials is pure titanium or titanium alloy because of its excellent mechanical properties, corrosion resistance, and biocompatibility. The direct bone bonding of titanium is known as osseointegration.1 However, some disadvantages of titanium implants, such as a dark grayish color2 which caused aesthetic problems by gingival recession, and metal sensitivity3,4 have also been observed. Siddiqi et al.5 reported that titanium may induce a hypersensitivity response in susceptible patients and could play an important role in the failure of titanium oral implants; nevertheless, little is known about titanium hypersensitivity. The poor machinability of titanium, galvanic corrosion, and plaque adhesion to titanium have also been reported as disadvantages.6–8
In recent years, the high-strength zirconia ceramic, yttria-stabilized tetragonal zirconia polycrystal (Y-TZP), has become an attractive new material for dental implants. YTZP provides high mechanical strength, fracture toughness, and biocompatibility. The high resistance of partially stabilized zirconia to crack propagation is based on phase transformation from the tetragonal to the monoclinic phase, leading to a volume expansion of approximately 3–4%.9–12 Esthetic performance can also be improved by using zirconia dental implants instead of titanium ones. Animal experiments evaluating bone-to-implant contact (BIC) or the push-in test using monkeys, dogs, or rats reported that Y-TZP implants were better or similar to titanium implants.13–16 In contrast, Hoffmann et al.17 found that zirconia implants had a slightly higher degree of bone apposition than that of titanium implants 2 weeks after implantation in the femoral condyles of rabbits; however, bone apposition was higher in titanium implants than in
Correspondence to: M. Hirota (e-mail: [email protected])
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zirconia ones at 4 weeks. A significant difference was also observed in the removal torque between titanium and zirconia implants at 12 weeks.18 To date, clear evidence showing osseointegration of partially stabilized zirconia has not yet been produced. Calcium phosphate coating on titanium implants, including hydroxyapatite coating, is a useful technique that improves and accelerates the bone healing process.19–21 The deposition of a carbonate-containing hydroxyapatite (CA) film is attractive because of its chemical resemblance to bone minerals. Sato et al.22 recently demonstrated that crystalline CA films could be deposited on titanium with a precursor solution of a calcium–EDTA complex and phosphate compounds, and this has been referred to as the molecular precursor method. Using this method, an apatite thin CA film with a thickness of less than 1 mm was firmly attached to titanium.23 An in vivo animal experiment demonstrated that the BIC of CA-coated titanium implants was greater than that of uncoated titanium implants.24 Kaneko et al.25 deposited a thin CA film on Y-TZP using the molecular precursor method and reported that initial cell adhesion of the mouse osteoblast-like cells MC3T3-E1 was enhanced on CA-coated Y-TZP, and the marked progression of actin filaments was also observed. Cell proliferation was also significantly higher in CA-coated Y-TZP than in YTZP. Based on the above-mentioned findings, we aimed to evaluate the biocompatibility of a thin coat of CA on Y-TZP using the molecular precursor method. An in vitro immersion experiment in simulated body fluid (SBF) and in vivo animal implantation experiment in the tibiae and femoral condyles of rabbits were performed. MATERIALS AND METHODS
Specimen preparation Yttria (3 mol %) stabilized tetragonal zirconia polycrystal (Y-TZP; TZ-3YB-E; Tosoh, Tokyo, Japan) was fabricated as a disk and cylinder. Y-TZP disks (12.0 mm in diameter and 1.0 mm in thickness) were used for characterization of the CA coating and the in vitro immersion experiment in SBF. Disk surfaces were polished with emery paper (]1200) under running water. Y-TZP cylinders (3.5 mm in diameter and 7.0 mm in height) were used for in vivo animal experiments. The cylindrical implant used in this study has a straight groove with the width of 1 mm on the head for fixing the implant into the bone. The surface was blasted and acid etched. Blasting was preformed perpendicularly to the surface from a distance of 20.0 mm with 150.0 lm alumina particles at 0.6 MPa air pressure, and acid etching was carried out on the blasted surface with 46% hydrofluoric acid (HF) for 15 min at room temperature. The disks and cylindrical specimens were then cleaned with an ultrasonic cleaner (VS-100III; AS ONE, Osaka, Japan) using ethanol and distilled water for 20 min. Apatite coating using the molecular precursor method Y-TZP disks and cylinders were coated with CA using the molecular precursor method previously described.23 The
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FIGURE 1. Preparation steps for the molecular precursor solution. Step 1: preparation of calcium–EDTA/amine ethanol solution. Step 2: preparation of the diphosphate salt. Step 3: to complete molecular precursor solution (Ca/P 5 1.67).
molecular precursor solution was prepared in three steps as shown in Figure 1. Briefly, step 1: preparation of a calcium– EDTA/amine ethanol solution, step 2: preparation of dibutylammonium diphosphate salt [((C4H9)2NH2)2P2O62H2O], and step 3: preparation of the molecular precursor solution by adding dibutylammonium diphosphate salt to calcium– EDTA/amine ethanol solution with an adjustment of Ca/P to 1.67 (Ca ion concentration 5 0.25 mmol/g). A molecular precursor solution of 25.0 lL was dropped on the Y-TZP disk surface and was spin coated with the double step mode at 500 rpm for 5 s and 2000 rpm for 30 s using a spin coater (1H-D7; MIKASA, Tokyo, Japan). A molecular precursor solution of 50.0 lL was dropped on the Y-TZP cylinder surface from the top to bottom in order to cover the entire area of the cylinder. The precursor films covered Y-TZP disks and cylinders were then heated at 600 C for 2 h using a tubular furnace (EPKPO12-K; ISUZU, Niigata, Japan) under oxygen gas introduction at a rate of 100 mL/min. A thin coating of CA could be achieved on YTZP (CA-Y-TZP) disks and cylinders using this method. Characterization of CA-coated films The surfaces of CA-Y-TZP disks were observed using a field emission-type scanning electron microscope (FE-SEM; JSM7001F, JEOL, Tokyo, Japan) after sputter coating with Au. Cross-sectional specimens were fabricated using a focus ion beam (FIB) system (Quanta 3D 200i; FEI, Tokyo, Japan) to observe the markedly thin CA films without inducing any damage. Processing of the observed part by a Ga ion beam was advanced in a general procedure according to a previously described technique.26,27 Cross-sectioned specimens were sputter coated with Au and were observed by FE-SEM. CA coating on Y-TZP was confirmed by energy dispersive X-ray spectroscopy (EDX, EMAX; HORIBA, Kyoto, Japan) at an accelerating voltage of 15 kV. The presence of the apatite coating was confirmed by elementary mapping of calcium and phosphorous. The crystal structure of the CA coating was characterized by X-ray diffraction (XRD; h22h diffractometer, MXP-18 AHF22; MAC Science, Kanagawa, Japan) with a thin layer attachment (incidence angle h 5 0.3 ), which had an X-ray source of Cu-Ka, power of 45 kV 3 300 mA, and Fourier transform infrared spectroscope (FT/IR-620; JASCO, Tokyo, Japan) at 4 cm21 resolution by attenuated total reflection.
BONE RESPONSES TO ZIRCONIA IMPLANTS WITH A THIN CA COATING
ORIGINAL RESEARCH REPORT
TABLE I. Concentrations of Electrolytes in HBSS 1
Ion
Na
Concentration (mmol/L)
142
K1
Mg21
Ca21
Cl2
HPO422
SO422
HCO2 3
5.81
0.811
1.26
145
0.778
0.811
4.17
Fourier transform infrared spectroscope (FT-IR) spectrum was obtained as a differential spectrum. First, the spectrum of Y-TZP was obtained and then infrared spectrum of CA-Y-TZP was subtracted from that of Y-TZP. Thereby, a differential spectrum was calculated. The number of samples for each characterization was 3, and three times measurements per sample were carried out for each characterization. Surface wettability and surface roughness Polished (#1200) and sandblasted–acid-etched (HF) disks were used for measurements. The surface wettability of the specimens was characterized by a contact angle measurement with respect to double distilled water using a Contact Angle Meter (Phoenix-ALPHA; Surface Electro Optics, Gyunggido, Korea). Five measurements of 15 s each were made for each surface type, and all analyses were performed at the same temperature and humidity. The surface roughness (Ra) of the specimens was measured with Handysurf E-30A (Tokyo Seimitsu, Tokyo, Japan) with a scan length of 4 mm and cutoff value of 0.8. Interference fringe was observed along the peripheral part of the disk due to the spin coat technique. Surface roughness was measured at the inner part of the disk. SBF immersion Hanks’ balanced salt solution (HBSS) without organic species was prepared as SBF.28 Concentrations of the used electrolytes for HBSS are listed in Table I. Y-TZP and CA-Y-TZP disks were immersed in 20.0 mL of HBSS with an adjusted pH to 7.4 at 37 C in a polypropylene bottle. The medium and bottles were replaced everyday to expose the disks to fresh medium. After immersion for 1, 3, 7, and 14 days, HBSS on the disks was removed using soft paper, and the disks were immediately dried in a desiccator. The surface appearances of Y-TZP and CA-Y-TZP disks after immersion in HBSS were observed using a scanning electron microscope (SEM, JSM-5600LV; JOEL, Tokyo, Japan) at an accelerating voltage of 15 kV. Specimens were sputter coated with Au before being examined. Cross-sectional views of the immersed samples were also performed. Specimens immersed in HBSS for 7 or 14 days in HBSS were embedded in epoxy resin (E-01-005; EPOCH, Tokyo, Japan). After curing the resin, samples were cut vertically by a microcutting machine (BS-300CP; EXAKT, Norderstedt, Germany) so that cross-sectional samples of the precipitates/Y-TZP or CA-Y-TZP could be obtained. Crosssectional samples were polished using emery paper of #1000 grade under running water. Samples were observed after ultrasonic cleaning using a SEM at an accelerating voltage of 15 kV. Specimens were sputter coated with Au before
being examined. The thickness of precipitated apatite layer was also measured. The crystallographic structure of precipitates on the YTZP and CA-Y-TZP disks was analyzed by XRD (Multi Flex 2kW; Rigaku, Tokyo, Japan), which had an X-ray source of Cu-Ka and power of 50 kV 3 50 mA, and was also determined by FT-IR (FTIR-8400S; Shimadzu Corp., Kyoto, Japan) using the KBr method. Precipitated products were detached from the Y-TZP and CA-Y-TZP disks for FT-IR measurements. Implantation procedure Before animal experiment, the presence of calcium and phosphorous of CA-Y-TZP cylinder was confirmed by electron probe micro-analysis (EPMA, JXA-8900R; JEOL) at an accelerating voltage of 20 kV by detecting the X-ray intensities of Ca-Ka and P-Ka. The specimens were embedded in epoxy resin. After curing the resin, the specimens were cut vertically through the middle using a cutting machine to observe the surface of the CA-Y-TZP implant. Then, carbon was coated onto the specimens before the EPMA analysis. The presence of apatite coating was confirmed by element mapping of calcium and phosphorus. The animal study was approved by the animal experimental ethical guidelines of Tsurumi University School of Dental Medicine (certificate no. 24A059). Six 10-week-old adult female Japanese white rabbits with weights ranging between 3.0 and 3.5 kg were used. Before surgery, Y-TZP and CA-Y-TZP cylinders were sterilized by ethylene oxide gas. Cylindrical implants were placed in the cortical bone of the left and right tibiae and also the trabecular bone of the left and right femoral condyles according to previous procedures.24,29 Each rabbit received Y-TZP and CA-Y-TZP implants (one in the left and the other in the right). A total of 12 implants were inserted, 6 Y-TZP and 6 CA-Y-TZP implants. Surgery was performed under general inhalation anesthesia with a 5% isoflurane and oxygen mixture, which was reduced to 2% isoflurane during surgical manipulation. Local anesthesia was performed by an injection of xylocaine. To reduce the risk of perioperative infection, a prophylactic antibiotic equivalent to latamoxef sodium (0.01 mg/kg Shiomalin; SHIONOGI, Osaka, Japan), was administered postoperatively by a subcutaneous injection. The hind legs of the rabbits were shaved, washed, and disinfected with iodine tincture. A longitudinal incision was made on the medial surface of the left and right tibiae and the bones were exposed by blunt dissection. A 2.0 mm pilot hole was drilled through the medial cortex of the tibia. A longitudinal incision for femoral implantation was made on the medial surface of the femur, and the medial condyle was exposed. After exposing the femoral condyle, a 2.0-mm pilot hole was drilled. The
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FIGURE 2. FE-SEM image of the Y-TZP and CA-Y-TZP disks (surface observation). (a) Y-TZP and (b) CA-Y-TZP.
hole was then gradually widened with a series of drills to the final diameter of the implant (3.5 mm). The bone defect was prepared with a very gentle surgical technique using a low rotational drilling speed (800 rpm) and continuous internal cooling. After press-fit insertion of the implants into bone defects in the tibiae or femoral condyles, the soft tissues were closed in separate layers using nonabsorbable sutures. Fluorochrome labeling was performed 11 weeks postsurgery with calcein (10 mg/kg, Calcein AM solution; DOJINDO LABORATORIES, Kumamoto, Japan). Rabbits were sacrificed 12 weeks after implantation by a peritoneal injection of an overdose of thiamylal sodium (Isothol; Nichi-Iko, Toyama, Japan). Histological and histomorphometrical observations The implants and surrounding bone were immediately excised and excess tissue was removed. After fixation in 10% buffered formalin solution, specimens were dehydrated through a graded series of ethanol and embedded in methylmethacrylate. Nondecalcified thin sections approximately 70 lm thick were made in a transverse direction perpendicular to the axis of the implants using a cutting-grinding technique (EXAKT-Cutting Grinding System, BS-300CP band system & 400CS microgrinding system; EXAKT Apparatebau, Norderstedt, Germany).30 Fluorochrome labeling by calcein was evaluated using a confocal laser scanning microscope (CLSM; TCS MultiPhoton, Leica, Germany) before staining. The size of the region of interest (ROI) was determined as a circle with a diameter of 110% of the inserted implant. The total length of calcein labels per area of the ROI on CLSM images was determined using an image analysis system (WinROOF, Visual System Division; Mitani Corporation, Tokyo, Japan). Sections were stained with methylene blue and basic fuchsin. The implant-bone interface was evaluated using a light microscope (Eclipse Ni; Nikon, Tokyo, Japan; magnification 3100). As well as a descriptive evaluation, histomorphometrical analysis was performed for new bone formation. Four or five sections were prepared from the same implant and one section was used for the histomor-
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phometrical evaluation. All measurements were taken from sections in the middle part of the implant. The percentage of BIC was determined. Measurements were performed along the total perimeter of the implant. The percentage of bone in the ROI around the implant was also measured and evaluated as bone mass (BM). BIC and BM were calculated using an image analysis system. Statistical analysis All data were calculated with the help of SPSS for Windows (SPSS Statics 17.0; SPSS, Chicago, USA). The contact angle measured, the Ra values, and the thickness of precipitated apatite layer in SBF immersions were compared using the Student t test. A p value 0.05). DISCUSSION
FIGURE 10. XRD patterns of deposited crystals on Y-TZP and CA-YTZP disks after 7 and 14 days of immersion in HBSS.
Experimental animals remained in good health during the test period. No clinical signs of inflammation or adverse tissue reactions were seen when animals were sacrificed and all cylinders were still in situ. Figures 13 and 14 show CLSM pictures of the Y-TZP and CA-Y-TZP cylinders in the tibia (cortical bone) and medial femoral condyle (trabecular bone), respectively. The green labeling inside the implants shown for Y-TZP was due to the presence of the straight groove on the head of the implanted Y-TZP cylinder, and the measurement of this part was excluded. Fluorochrome green labeling by the administration of calcein was clearly observed around the implants 11 weeks postsurgery. The intensity of green labeling in the tibia and femoral condyle for the Y-TZP implants (a) was stronger than that for the CA-Y-TZP implants (b). The lengths of fluorescence labeling by calcein in the ROI, which was representative of the new bone formation rate, are shown in Table III. The length of fluorescence labeling in the Y-TZP implants was significantly higher than that of the CA-Y-TZP implants in the tibia (p < 0.05). No significant difference was observed in the femoral condyle between Y-TZP and CA-Y-TZP (p > 0.05). The histological appearances of the Y-TZP and CA-Y-TZP implants in the tibia (cortical bone) and medial femoral condyle (trabecular bone) after 12 weeks of implantation are shown in Figures 15 and 16, respectively. New bone formation was observed surrounding implant materials. Remodeling and compaction of the bone–implant interface were complete for both tibial and femoral implants, No signs of an inflammatory response were detected in the tissue surrounding the implants. Tighter BIC was observed for CA-YTZP implants not only tibial but also femoral implantation. The presence of fibrous tissue [Figure 15(a)] and bone marrow [Figure 16(a)] was identified for Y-TZP implants. Newly formed interfacial bone for the tibial implant had a lamellar appearance and more interfacial bone contact was observed with CA-Y-TZP than with Y-TZP. New bone had completely remodeled into mature trabecular bone for the femoral implant. Bone marrow tissue was present between the areas of bone contact. All implant surfaces were partially covered with new bone. CA-Y-TZP showed more bone contact than that of Y-TZP.
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In this study, the biocompatibility of a thin CA-coated Y-TZP, using the molecular precursor method, was evaluated by in vitro SBF immersion experiments and in vivo animal implantation experiments. Cylindrical implants were inserted into the tibiae and femoral condyles of rabbits. Several methods have been proposed for apatite coating. A very thin apatite coating technique such as magnetron sputtering was previously developed to overcome the disadvantages associated with the plasma spray method.29,31 The molecular precursor method is a new wet process for coating thin CA layers onto titanium. The advantage of the molecular precursor method is its simplicity and also that the CA coating can be deposited onto substrates of any shape. The inside of a three-dimensional titanium fiber mesh was previously shown to be coated with a thin CA film using the molecular precursor method.32,33 The interface between the deposited CA film and Y-TZP was observed using a FIB processing technique, which can produce nano-scale specimens without any damage. Tanaka27 reported the structures of two types of titanium oxide interfaces using the FIB technique. A cross-sectional view of CA-YTZP revealed a uniform and firmly adhered CA film on Y-TZP with a thickness of less than 1 mm. A difference in CA film thickness was observed between the inner side and outer side of the disk. The spin coat technique was used in this study to achieve a thin CA coating with the molecular precursor method. We speculated that easy diffusion of the precursor solution toward the outer direction of the disk produced a difference in the thickness of the CA film. XRD and FT-IR measurements were used to characterize CA films, which was consistent with a previous report.24 EDX measurements also identified the Ca and P atoms of the CA coating on the surface of CA-Y-TZP, indicating the uniform coating of thin CA layer.
FIGURE 11. FT-IR spectrum of deposited crystals on Y-TZP and CA-YTZP disks after 7 and 14 days of immersion in HBSS.
BONE RESPONSES TO ZIRCONIA IMPLANTS WITH A THIN CA COATING
FIGURE 12. EPMA analysis of CA film on Y-TZP cylinder. Zr: zirconium, Ca: calcium, and P: phosphorus.
FIGURE 13. CLSM pictures of Y-TZP and CA-Y-TZP cylinders in the tibia (cortical bone) in which fluorochrome labeling was performed with calcein 11 weeks postsurgery. (a) Y-TZP and (b) CA-Y-TZP.
FIGURE 14. CLSM pictures of Y-TZP and CA-Y-TZP cylinders in femoral condyle (trabecular bone) in which fluorochrome labeling was performed with calcein 11 weeks postsurgery. (a) Y-TZP and (b) CA-Y-TZP.
In the SBF immersion experiments, apatite deposition on the CA-Y-TZP disks had progressed more than that on the YTZP disks. To evaluate in vitro biocompatibility, many studies have reported the formation of an apatite layer after materials were immersed in SBF. In this study, HBSS was used as SBF. Hanawa and Ota28 reported that an apatite layer formed on a titanium surface after immersion in HBSS. The in vivo bioactivity (osteoconductivity) of biomaterials such as ceramics was shown to precisely mirror their in vitro apatite-forming ability in SBF,34 namely, the more apatite that formed, the better bone formation was. In vivo bone formation on a CA-coated titanium fiber mesh, reported by Hayakawa et al.,32 confirmed the results of the in vitro SBF immersion experiment. Thus, CA-Y-TZP was expected to enhance bone formation in vivo. In the animal experiments, Y-TZP and CA-Y-TZP cylinders were sand-blasted with large grits and acid etched with HF. This surface treatment was similar to the process known as SLA for titanium implants.35 In consideration of future clinical applications, the above-mentioned surface treatment was applied to cylindrical implants. In this study, uniform CA coating could be deposited on cylindrical shape of Y-TZP by using molecular precursor method. In this study, implants were inserted into cortical tibia bone and trabecular femoral condyle bone. Cortical bone is known to be denser and stiffer than trabecular bone. The elastic modulus of cortical bone was previously shown to be higher than that of trabecular bone.36 The implant site TABLE III. Length of Fluorescence Labeling Length of Fluorescence Labeling (mm) Specimen Y-TZP CA-Y-TZP
Tibia
Femoral Condyle a
10.9 (0.3) 7.9 (0.7)a
Values in brackets are SD. a Significantly different at p < 0.05.
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9.8 (3.1) 6.4 (1.8)
between cortical and trabecular bone has been shown to influence the bone response to implants.37 Hayakawa et al.29 reported that cortical and trabecular bone exhibited different bone responses toward apatite-coated titanium implants. Fluorochrome labeling by calcein demonstrated new bone formation in the surrounding areas of the implants. Fluorescent dyes chelate to calcium ions, resulting in the deposition of a multiple vital label on all actively mineralizing bone surfaces.38 The labeling length for CA-Y-TZP was significant less in the tibia, which corresponded to the lower activity of new bone formation by CA-Y-TZP than by Y-TZP. However, no significant difference was observed in BM around the implants. The maturation of woven bone for CAY-TZP may have occurred at an earlier stage of the bone healing process than that for Y-TZP. The BIC and BM for CA-Y-TZP were significantly higher in the femoral condyle. Our present results for in vivo apatite formation in SBF corresponded with the in vivo bone response. This study revealed that coating CA on Y-TZP using the molecular precursor method promoted new trabecular bone formation surrounding the implants. This was due to improved osteoconductivity by the thin CA coating. The present results indicate that the CA-Y-TZP implant may be suitable for a maxillary site with poor bone quality; however, the length of stability should be confirmed in future studies. No significant difference was observed in BIC and BM between Y-TZP and CA-Y-TZP for cortical bone in the tibia. Differences in the bone response to the Y-TZP implant between cortical bone and trabecular bone may lead to different healing mechanisms, that is, direct bone apposition in tibial cortical bone and endochondral bone formation in the femoral cortical bone. More detailed studies are needed to clarify these different bone responses. Albrektsson et al.39 suggested that osseointegration corresponded to approximately 60% bone contact for titanium
BONE RESPONSES TO ZIRCONIA IMPLANTS WITH A THIN CA COATING
ORIGINAL RESEARCH REPORT
FIGURE 15. Histological appearance of Y-TZP and CA-Y-TZP cylinders in the tibia (cortical bone) after 12 weeks of implantation. (methylene blue and basic fuchsin; original magnification 3100). (a) Y-TZP and (b) CA-Y-TZP.
FIGURE 16. Histological appearance of Y-TZP and CA-Y-TZP cylinders in the femoral condyle (trabecular bone) after 12 weeks of implantation. (methylene blue and basic fuchsin; original magnification 3100). (a) Y-TZP and (b) CA-Y-TZP.
implants. The BIC of the Y-TZP implant in this study was approximately 40–60%. The BIC of the CA-Y-TZP implant for tibial cortical bone was approximately 80%. In contrast, the BIC of the CA-Y-TZP implant for the trabecular femoral condyle was above the limit proposed by Albrestsson et al., but was lower than that of titanium. For example, Hayakawa et al.24 reported that CA-coated titanium implants using the molecular precursor method had a BIC of 70–80% after 12 weeks of implantation into the trabecular bone of rabbit. To improve the bone response, a more detailed study on the thin CA coating, such as the thickness of the CA film or calTABLE IV. Percentage of the Measured BIC and BM BIC (%)
BM (%)
Specimen
Tibia
Femoral Condyle
Tibia
Femoral Condyle
Y-TZP CA-Y-TZP
58.0 (13.8) 79.2 (1.2)
46.1 (4.7)a 67.6 (4.4)a
85.3 (8.8) 96.4 (1.8)
65.2 (10.0)b 79.1 (6.92)b
Values in brackets are SD. a,b Significantly different at p < 0.05.
cium concentration in the molecular precursor solution, is needed. CONCLUSIONS
A thin and uniform CA coating could be deposited on Y-TZP using the molecular precursor method. In the SBF immersion experiment, apatite deposition appeared to have progressed more on the CA-Y-TZP cylinder than on the Y-TZP cylinder. A larger amount of new bone formation was demonstrated in animal experiments and a significantly higher BIC ratio was observed in the trabecular femora condyle. We suggest that thin CA-coated Y-TZP using the molecular precursor method may be applicable as an innovative metal-free implant. ACKNOWLEDGMENTS
We acknowledge Professor Masao Yoshinari, Tokyo Dental College, for his help for the preparation of Y-TZP disk and cylindrical samples and Dr. Kouji Inoue, Tsurumi University School of Dental Medicine, for his help for SEM and EPMA analysis.
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REFERENCES 1. Bra˚nemark PI. Osseointegration and its experimental background. J Prosthet Dent 1983;50:399–410. €chle M, Strub JR. Loaded custom-made zir2. Kohal RJ, Weng D, Ba conia and titanium implants show similar osseointegration: An animal experiment. J Periodontol 2004;75:1262–1268. 3. Yamauchi R, Morita A, Tsuji T. Pacemaker dermatitis from titanium. Contact Dermatitis 2000;42:52–53. 4. Valentine-Thon E, Schiwara HW. Validity of MELISA for metal sensitivity testing. Neuro Endocrinol Lett 2003;24:57–64. 5. Siddiqi A, Payne AG, De Silva RK, Duncan WJ. Titanium allergy: Could it affect dental implant integration? Clin Oral Implants Res 2011;22:673–680. 6. Koike M, Jacobson D, Chan KS, Okabe T. Grindability alpha-case formed on cast titanium. Dent Mater J 2009;28(5):587–594. 7. Chaturvedi TP. An overview of the corrosion aspect of dental implants (titanium and its alloys). Indian J Dent Res 2009;20:91–98. 8. Yamane K, Ayukawa Y, Takeshita T, Furuhashi A, Yamashita Y, Koyano K. Bacterial adhesion affinities of various implant abutment materials. Clin Oral Implants Res 2012;00:1–6. 9. Garvie RC, Hannink RH, Pascoe RT. Ceramic steel? Nature 1975; 258:703–704. 10. Lange FF. Transformation toughening: Part 3. Experimental observations in the ZrO2-Y2O3 system. J Mater Sci 1982;17:240–246. 11. Evans AG. Perspective on the development of high-toughness ceramics. J Am Ceram Soc 1990;73:187–206. 12. Piconi C, Maccauro G. Zirconia as a ceramic biomaterial. Biomaterials 1999;20:1–25. 13. Akagawa Y, Ichikawa Y, Nikai H, Tsuru H. Interface histology of unloaded and early loaded partially stabilized zirconia endosseous implant in initial bone healing. J Prosthet Dent 1993;69:599–604. 14. Akagawa Y, Hosokawa R, Sato Y, Kamayama K. Comparison between freestanding and tooth-connected partially stabilized zirconia implants after two years’ function in monkeys: A clinical and histologic study. J Prosthet Dent 1998;80:551–558. 15. Andreiotelli M, Wenz HJ, Kohal RJ. Are ceramic implants a viable alternative to titanium implants? A systematic literature review. Clin Oral Implants Res 2009;20:32–47. € lu E. Zirconia dental implants: A literature 16. Ozkurt Z, Kazazog review. J Oral Implantol 2011;37(3):367–376. 17. Hoffmann O, Angelov N, Gallez F, Jung RE, Weber FE. The zirconia implant–bone interface: A preliminary histologic evaluation in rabbits. Int J Oral Maxillofac Implants 2008;23:691–695. 18. Hoffmann O, Angelov N, Zafiropoulos GG, Andreana S. Osseointegration of zirconia implants with different surface characteristics: An evaluation in rabbits. Int J Oral Maxillofac Implants 2012; 27:352–358. 19. Ong JL, Chan DC. Hydroxyapatite and their use as coatings in dental implants: A review. Crit Rev Biomed Eng 2000;28:667–707. 20. de Jonge LT, Leeuwenburgh SC, Wolke JG, Jansen JA. Organic– inorganic surface modifications for titanium implant surfaces. Pharm Res 2008;25:2357–2369. 21. Wennerberg A, Albrektsson T. Structural influence from calcium phosphate coating and its possible effect on enhanced bone integration. Acta Odontol Scand 2009;67:333–340. 22. Sato M, Hara H, Tomikoshi H, Mochizuki C. Film fabrication of monolithic hydroxyapatite by an application of molecular precursor method. In: Proceeding of International Symposium on Advanced Materials (ISAM/ISAT2); 2003. p 892.
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23. Takahashi K, Hayakawa T, Yoshinari M, Hara H, Mochizuki C, Sato M, Nemoto K. Molecular precursor method for thin calcium phosphate coating on titanium. Thin solid films 2005;484:1–9. 24. Hayakawa T, Takahashi K, Yoshinari M, Okada H, Yamamoto H, Sato M, Nemoto K. Trabecular bone response to titanium implants with a thin carbonate-containing apatite coating applied using the molecular precursor method. Int J Oral Maxillofac Implants 2006;21:851–858. 25. Kaneko H, Sasaki H, Honma S, Hayakawa T, Sato M, Yajima Y, Yoshinari M. Influence of thin carbonate-containing apatite coating with molecular precursor method to zirconia on osteoblastlike cell response. Dent Mater J 2014;33:39–47. 26. Ngo H, Cairney J, Munroe P, Vargas M, Mount G. Focused ion beam in dental research. Am J Dent 2000;13:31–34. 27. Tanaka Y. Titanium-oxide interface structures formed by degassing and anodization processes. J Mater Sci 2005;40:3081–3090. 28. Hanawa T, Ota M. Calcium phosphate naturally formed on titanium in electrolyte solution. Biomaterials 1991;12:767–774. 29. Hayakawa T, Yoshinari M, Nemoto K, Wolke JGC, Jansen JA. Effect of surface roughness and calcium phosphate coating on the implant/bone response. Clin Oral Implants Res 2000;11:296– 304. 30. Donath K, Breuner G. A method for study of undecalcified bones and teeth with attached soft tissues: The Sage-Schliff (sawing and grinding) technique. J Oral Pathol 1982;11:318–326. 31. Hulshoff JE, Hayakawa T, van Dijk K, Leijdekkers-Govers AF, van der Waerden JP, Jansen JA. Mechanical and histologic evaluation of Ca–P plasma-spray and magnetron sputter-coated implants in trabecular bone of the goat. J Biomed Mater Res 1997;36:75–83. 32. Hayakawa T, Takahashi K, Okada H, Yoshinari M, Hara H, Mochizuki C, Yamamoto H, Sato M. Effect of thin carbonatecontaining apatite (CA) coating fiber mesh on trabecular bone response. J Mater Sci Mater Med 2008;19:2087–2096. 33. Hirota M, Hayakawa T, Ametani A, Kuboki Y, Sato M, Tohnai I. The effect of hydroxyapatite-coated titanium fiber web on human osteoblast functional activity. Int J Oral Maxillofac Implants 2011; 26:245–250. 34. Kokubo T, Takamada H. How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 2006;27:2907–2915. €gger U, Ramseier CA, Salvi 35. Buser D, Janner SF, Wittneben JG, Bra GE. 10-year survival and success rates of 511 titanium implants with a sandblasted and acid-etched surface: A retrospective study in 303 partially edentulous patients. Clin Implant Dent Relat Res 2012;14:839–851. 36. Seong WJ, Kim UK, Swift JQ, Heo YC, Hodges JS, Ko CC. Elastic properties and apparent density of human edentulous maxilla and mandible. Int J Oral Maxillofac Surg 2009;38:1088–1093. € ger M, Neukam FW, 37. Nkenke E, Hahn M, Weinzieri K, Radespiel-Tro Engelke K. Implant stability and histomorphometry: A correlation study in human cadavers using stepped cylinder implants. Clin Oral Implants Res 2003;14:601–609. € nig J 38. Ko unior B, Beck TJ, Kappert HF, Kappert CC, Masuko TS. A study of different calcification areas in newly formed bone 8 weeks after insertion of dental implants in rabbit tibias. Ann Anat 1998;180:471–475. 39. Albrektsson T, Eriksson AR, Friberg B, Lekholm U, Lindahl L, Nevins M, Oikarinen V, Roos J, Sennerby L, Astrand P. Histologic investigations on 33 retrieved Nobelpharma implants. Clin Mater 1993;12:1–9.
BONE RESPONSES TO ZIRCONIA IMPLANTS WITH A THIN CA COATING
Department of Removable Prosthodontics, Tsurumi University School of Dental Medicine, Kanagawa, Japan Department of Dental Engineering, Tsurumi University School of Dental Medicine, Kanagawa, Japan 3 Division of Liberal Arts, Kogakuin University, Tokyo, Japan 4 Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, Tokyo, Japan 5 Engineering Materials Science, Department of Advance Materials Science, Faculty of Engineering, Kagawa University, Kagawa, Japan 2
Received 17 September 2013; revised 16 December 2013; accepted 7 January 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33112 Abstract: Thin carbonate-containing hydroxyapatite (CA) films coating partially stabilized zirconia (Y-TZP) were prepared (CA-Y-TZP) to establish a metal-free implant system. CA was coated using a molecular precursor method. The CA film was deposited on the surface of Y-TZP using a precursor solution, which was a mixture of a calcium–ethylenediaminetetraacetic acid (EDTA) complex and phosphate compounds. The deposited CA film was characterized by X-ray diffraction, Fourier transform infrared spectroscopy, and energy dispersive X-ray spectroscopy measurements. A focus ion beam system technique revealed that the thickness of the CA film was less than 1.0 mm. Biological evaluations of CA-Y-TZP were performed by immersion experiments in simulated body fluid (SBF) and implantation experiments in the tibiae
and femoral condyles of rabbits. In the SBF immersion experiment, apatite deposition progressed more on CA-Y-TZP at the early stage of immersion than on Y-TZP without the CA coating. Animal experiments revealed that bone formation on CA-Y-TZP was similar with than on Y-TZP. Histomorphometrical evaluations showed a significantly higher boneto-implant contact ratio and bone mass on CA-Y-TZP after implantation into the femoral trabecular bone of rabbits. Therefore, CA-Y-TZP appears to be applicable as a metal-free C 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: implant. V Appl Biomater 00B:000–000, 2014.
Key Words: dental implants, partially stabilized zirconia, apatite coating, simulated body fluid, bone, implant contact
How to cite this article: Hirota M, Hayakawa T, Ohkubo C, Sato M, Hara H, Toyama T, Tanaka Y. 2014. Bone responses to zirconia implants with a thin carbonate-containing hydroxyapatite coating using a molecular precursor method. J Biomed Mater Res Part B 2014:00B:000–000.
INTRODUCTION
Dental implants have been extensively used for fixed, removable, and maxillofacial prosthodontic rehabilitation with a sufficient success rate. The first choice of implant materials is pure titanium or titanium alloy because of its excellent mechanical properties, corrosion resistance, and biocompatibility. The direct bone bonding of titanium is known as osseointegration.1 However, some disadvantages of titanium implants, such as a dark grayish color2 which caused aesthetic problems by gingival recession, and metal sensitivity3,4 have also been observed. Siddiqi et al.5 reported that titanium may induce a hypersensitivity response in susceptible patients and could play an important role in the failure of titanium oral implants; nevertheless, little is known about titanium hypersensitivity. The poor machinability of titanium, galvanic corrosion, and plaque adhesion to titanium have also been reported as disadvantages.6–8
In recent years, the high-strength zirconia ceramic, yttria-stabilized tetragonal zirconia polycrystal (Y-TZP), has become an attractive new material for dental implants. YTZP provides high mechanical strength, fracture toughness, and biocompatibility. The high resistance of partially stabilized zirconia to crack propagation is based on phase transformation from the tetragonal to the monoclinic phase, leading to a volume expansion of approximately 3–4%.9–12 Esthetic performance can also be improved by using zirconia dental implants instead of titanium ones. Animal experiments evaluating bone-to-implant contact (BIC) or the push-in test using monkeys, dogs, or rats reported that Y-TZP implants were better or similar to titanium implants.13–16 In contrast, Hoffmann et al.17 found that zirconia implants had a slightly higher degree of bone apposition than that of titanium implants 2 weeks after implantation in the femoral condyles of rabbits; however, bone apposition was higher in titanium implants than in
Correspondence to: M. Hirota (e-mail: [email protected])
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zirconia ones at 4 weeks. A significant difference was also observed in the removal torque between titanium and zirconia implants at 12 weeks.18 To date, clear evidence showing osseointegration of partially stabilized zirconia has not yet been produced. Calcium phosphate coating on titanium implants, including hydroxyapatite coating, is a useful technique that improves and accelerates the bone healing process.19–21 The deposition of a carbonate-containing hydroxyapatite (CA) film is attractive because of its chemical resemblance to bone minerals. Sato et al.22 recently demonstrated that crystalline CA films could be deposited on titanium with a precursor solution of a calcium–EDTA complex and phosphate compounds, and this has been referred to as the molecular precursor method. Using this method, an apatite thin CA film with a thickness of less than 1 mm was firmly attached to titanium.23 An in vivo animal experiment demonstrated that the BIC of CA-coated titanium implants was greater than that of uncoated titanium implants.24 Kaneko et al.25 deposited a thin CA film on Y-TZP using the molecular precursor method and reported that initial cell adhesion of the mouse osteoblast-like cells MC3T3-E1 was enhanced on CA-coated Y-TZP, and the marked progression of actin filaments was also observed. Cell proliferation was also significantly higher in CA-coated Y-TZP than in YTZP. Based on the above-mentioned findings, we aimed to evaluate the biocompatibility of a thin coat of CA on Y-TZP using the molecular precursor method. An in vitro immersion experiment in simulated body fluid (SBF) and in vivo animal implantation experiment in the tibiae and femoral condyles of rabbits were performed. MATERIALS AND METHODS
Specimen preparation Yttria (3 mol %) stabilized tetragonal zirconia polycrystal (Y-TZP; TZ-3YB-E; Tosoh, Tokyo, Japan) was fabricated as a disk and cylinder. Y-TZP disks (12.0 mm in diameter and 1.0 mm in thickness) were used for characterization of the CA coating and the in vitro immersion experiment in SBF. Disk surfaces were polished with emery paper (]1200) under running water. Y-TZP cylinders (3.5 mm in diameter and 7.0 mm in height) were used for in vivo animal experiments. The cylindrical implant used in this study has a straight groove with the width of 1 mm on the head for fixing the implant into the bone. The surface was blasted and acid etched. Blasting was preformed perpendicularly to the surface from a distance of 20.0 mm with 150.0 lm alumina particles at 0.6 MPa air pressure, and acid etching was carried out on the blasted surface with 46% hydrofluoric acid (HF) for 15 min at room temperature. The disks and cylindrical specimens were then cleaned with an ultrasonic cleaner (VS-100III; AS ONE, Osaka, Japan) using ethanol and distilled water for 20 min. Apatite coating using the molecular precursor method Y-TZP disks and cylinders were coated with CA using the molecular precursor method previously described.23 The
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FIGURE 1. Preparation steps for the molecular precursor solution. Step 1: preparation of calcium–EDTA/amine ethanol solution. Step 2: preparation of the diphosphate salt. Step 3: to complete molecular precursor solution (Ca/P 5 1.67).
molecular precursor solution was prepared in three steps as shown in Figure 1. Briefly, step 1: preparation of a calcium– EDTA/amine ethanol solution, step 2: preparation of dibutylammonium diphosphate salt [((C4H9)2NH2)2P2O62H2O], and step 3: preparation of the molecular precursor solution by adding dibutylammonium diphosphate salt to calcium– EDTA/amine ethanol solution with an adjustment of Ca/P to 1.67 (Ca ion concentration 5 0.25 mmol/g). A molecular precursor solution of 25.0 lL was dropped on the Y-TZP disk surface and was spin coated with the double step mode at 500 rpm for 5 s and 2000 rpm for 30 s using a spin coater (1H-D7; MIKASA, Tokyo, Japan). A molecular precursor solution of 50.0 lL was dropped on the Y-TZP cylinder surface from the top to bottom in order to cover the entire area of the cylinder. The precursor films covered Y-TZP disks and cylinders were then heated at 600 C for 2 h using a tubular furnace (EPKPO12-K; ISUZU, Niigata, Japan) under oxygen gas introduction at a rate of 100 mL/min. A thin coating of CA could be achieved on YTZP (CA-Y-TZP) disks and cylinders using this method. Characterization of CA-coated films The surfaces of CA-Y-TZP disks were observed using a field emission-type scanning electron microscope (FE-SEM; JSM7001F, JEOL, Tokyo, Japan) after sputter coating with Au. Cross-sectional specimens were fabricated using a focus ion beam (FIB) system (Quanta 3D 200i; FEI, Tokyo, Japan) to observe the markedly thin CA films without inducing any damage. Processing of the observed part by a Ga ion beam was advanced in a general procedure according to a previously described technique.26,27 Cross-sectioned specimens were sputter coated with Au and were observed by FE-SEM. CA coating on Y-TZP was confirmed by energy dispersive X-ray spectroscopy (EDX, EMAX; HORIBA, Kyoto, Japan) at an accelerating voltage of 15 kV. The presence of the apatite coating was confirmed by elementary mapping of calcium and phosphorous. The crystal structure of the CA coating was characterized by X-ray diffraction (XRD; h22h diffractometer, MXP-18 AHF22; MAC Science, Kanagawa, Japan) with a thin layer attachment (incidence angle h 5 0.3 ), which had an X-ray source of Cu-Ka, power of 45 kV 3 300 mA, and Fourier transform infrared spectroscope (FT/IR-620; JASCO, Tokyo, Japan) at 4 cm21 resolution by attenuated total reflection.
BONE RESPONSES TO ZIRCONIA IMPLANTS WITH A THIN CA COATING
ORIGINAL RESEARCH REPORT
TABLE I. Concentrations of Electrolytes in HBSS 1
Ion
Na
Concentration (mmol/L)
142
K1
Mg21
Ca21
Cl2
HPO422
SO422
HCO2 3
5.81
0.811
1.26
145
0.778
0.811
4.17
Fourier transform infrared spectroscope (FT-IR) spectrum was obtained as a differential spectrum. First, the spectrum of Y-TZP was obtained and then infrared spectrum of CA-Y-TZP was subtracted from that of Y-TZP. Thereby, a differential spectrum was calculated. The number of samples for each characterization was 3, and three times measurements per sample were carried out for each characterization. Surface wettability and surface roughness Polished (#1200) and sandblasted–acid-etched (HF) disks were used for measurements. The surface wettability of the specimens was characterized by a contact angle measurement with respect to double distilled water using a Contact Angle Meter (Phoenix-ALPHA; Surface Electro Optics, Gyunggido, Korea). Five measurements of 15 s each were made for each surface type, and all analyses were performed at the same temperature and humidity. The surface roughness (Ra) of the specimens was measured with Handysurf E-30A (Tokyo Seimitsu, Tokyo, Japan) with a scan length of 4 mm and cutoff value of 0.8. Interference fringe was observed along the peripheral part of the disk due to the spin coat technique. Surface roughness was measured at the inner part of the disk. SBF immersion Hanks’ balanced salt solution (HBSS) without organic species was prepared as SBF.28 Concentrations of the used electrolytes for HBSS are listed in Table I. Y-TZP and CA-Y-TZP disks were immersed in 20.0 mL of HBSS with an adjusted pH to 7.4 at 37 C in a polypropylene bottle. The medium and bottles were replaced everyday to expose the disks to fresh medium. After immersion for 1, 3, 7, and 14 days, HBSS on the disks was removed using soft paper, and the disks were immediately dried in a desiccator. The surface appearances of Y-TZP and CA-Y-TZP disks after immersion in HBSS were observed using a scanning electron microscope (SEM, JSM-5600LV; JOEL, Tokyo, Japan) at an accelerating voltage of 15 kV. Specimens were sputter coated with Au before being examined. Cross-sectional views of the immersed samples were also performed. Specimens immersed in HBSS for 7 or 14 days in HBSS were embedded in epoxy resin (E-01-005; EPOCH, Tokyo, Japan). After curing the resin, samples were cut vertically by a microcutting machine (BS-300CP; EXAKT, Norderstedt, Germany) so that cross-sectional samples of the precipitates/Y-TZP or CA-Y-TZP could be obtained. Crosssectional samples were polished using emery paper of #1000 grade under running water. Samples were observed after ultrasonic cleaning using a SEM at an accelerating voltage of 15 kV. Specimens were sputter coated with Au before
being examined. The thickness of precipitated apatite layer was also measured. The crystallographic structure of precipitates on the YTZP and CA-Y-TZP disks was analyzed by XRD (Multi Flex 2kW; Rigaku, Tokyo, Japan), which had an X-ray source of Cu-Ka and power of 50 kV 3 50 mA, and was also determined by FT-IR (FTIR-8400S; Shimadzu Corp., Kyoto, Japan) using the KBr method. Precipitated products were detached from the Y-TZP and CA-Y-TZP disks for FT-IR measurements. Implantation procedure Before animal experiment, the presence of calcium and phosphorous of CA-Y-TZP cylinder was confirmed by electron probe micro-analysis (EPMA, JXA-8900R; JEOL) at an accelerating voltage of 20 kV by detecting the X-ray intensities of Ca-Ka and P-Ka. The specimens were embedded in epoxy resin. After curing the resin, the specimens were cut vertically through the middle using a cutting machine to observe the surface of the CA-Y-TZP implant. Then, carbon was coated onto the specimens before the EPMA analysis. The presence of apatite coating was confirmed by element mapping of calcium and phosphorus. The animal study was approved by the animal experimental ethical guidelines of Tsurumi University School of Dental Medicine (certificate no. 24A059). Six 10-week-old adult female Japanese white rabbits with weights ranging between 3.0 and 3.5 kg were used. Before surgery, Y-TZP and CA-Y-TZP cylinders were sterilized by ethylene oxide gas. Cylindrical implants were placed in the cortical bone of the left and right tibiae and also the trabecular bone of the left and right femoral condyles according to previous procedures.24,29 Each rabbit received Y-TZP and CA-Y-TZP implants (one in the left and the other in the right). A total of 12 implants were inserted, 6 Y-TZP and 6 CA-Y-TZP implants. Surgery was performed under general inhalation anesthesia with a 5% isoflurane and oxygen mixture, which was reduced to 2% isoflurane during surgical manipulation. Local anesthesia was performed by an injection of xylocaine. To reduce the risk of perioperative infection, a prophylactic antibiotic equivalent to latamoxef sodium (0.01 mg/kg Shiomalin; SHIONOGI, Osaka, Japan), was administered postoperatively by a subcutaneous injection. The hind legs of the rabbits were shaved, washed, and disinfected with iodine tincture. A longitudinal incision was made on the medial surface of the left and right tibiae and the bones were exposed by blunt dissection. A 2.0 mm pilot hole was drilled through the medial cortex of the tibia. A longitudinal incision for femoral implantation was made on the medial surface of the femur, and the medial condyle was exposed. After exposing the femoral condyle, a 2.0-mm pilot hole was drilled. The
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FIGURE 2. FE-SEM image of the Y-TZP and CA-Y-TZP disks (surface observation). (a) Y-TZP and (b) CA-Y-TZP.
hole was then gradually widened with a series of drills to the final diameter of the implant (3.5 mm). The bone defect was prepared with a very gentle surgical technique using a low rotational drilling speed (800 rpm) and continuous internal cooling. After press-fit insertion of the implants into bone defects in the tibiae or femoral condyles, the soft tissues were closed in separate layers using nonabsorbable sutures. Fluorochrome labeling was performed 11 weeks postsurgery with calcein (10 mg/kg, Calcein AM solution; DOJINDO LABORATORIES, Kumamoto, Japan). Rabbits were sacrificed 12 weeks after implantation by a peritoneal injection of an overdose of thiamylal sodium (Isothol; Nichi-Iko, Toyama, Japan). Histological and histomorphometrical observations The implants and surrounding bone were immediately excised and excess tissue was removed. After fixation in 10% buffered formalin solution, specimens were dehydrated through a graded series of ethanol and embedded in methylmethacrylate. Nondecalcified thin sections approximately 70 lm thick were made in a transverse direction perpendicular to the axis of the implants using a cutting-grinding technique (EXAKT-Cutting Grinding System, BS-300CP band system & 400CS microgrinding system; EXAKT Apparatebau, Norderstedt, Germany).30 Fluorochrome labeling by calcein was evaluated using a confocal laser scanning microscope (CLSM; TCS MultiPhoton, Leica, Germany) before staining. The size of the region of interest (ROI) was determined as a circle with a diameter of 110% of the inserted implant. The total length of calcein labels per area of the ROI on CLSM images was determined using an image analysis system (WinROOF, Visual System Division; Mitani Corporation, Tokyo, Japan). Sections were stained with methylene blue and basic fuchsin. The implant-bone interface was evaluated using a light microscope (Eclipse Ni; Nikon, Tokyo, Japan; magnification 3100). As well as a descriptive evaluation, histomorphometrical analysis was performed for new bone formation. Four or five sections were prepared from the same implant and one section was used for the histomor-
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phometrical evaluation. All measurements were taken from sections in the middle part of the implant. The percentage of BIC was determined. Measurements were performed along the total perimeter of the implant. The percentage of bone in the ROI around the implant was also measured and evaluated as bone mass (BM). BIC and BM were calculated using an image analysis system. Statistical analysis All data were calculated with the help of SPSS for Windows (SPSS Statics 17.0; SPSS, Chicago, USA). The contact angle measured, the Ra values, and the thickness of precipitated apatite layer in SBF immersions were compared using the Student t test. A p value 0.05). DISCUSSION
FIGURE 10. XRD patterns of deposited crystals on Y-TZP and CA-YTZP disks after 7 and 14 days of immersion in HBSS.
Experimental animals remained in good health during the test period. No clinical signs of inflammation or adverse tissue reactions were seen when animals were sacrificed and all cylinders were still in situ. Figures 13 and 14 show CLSM pictures of the Y-TZP and CA-Y-TZP cylinders in the tibia (cortical bone) and medial femoral condyle (trabecular bone), respectively. The green labeling inside the implants shown for Y-TZP was due to the presence of the straight groove on the head of the implanted Y-TZP cylinder, and the measurement of this part was excluded. Fluorochrome green labeling by the administration of calcein was clearly observed around the implants 11 weeks postsurgery. The intensity of green labeling in the tibia and femoral condyle for the Y-TZP implants (a) was stronger than that for the CA-Y-TZP implants (b). The lengths of fluorescence labeling by calcein in the ROI, which was representative of the new bone formation rate, are shown in Table III. The length of fluorescence labeling in the Y-TZP implants was significantly higher than that of the CA-Y-TZP implants in the tibia (p < 0.05). No significant difference was observed in the femoral condyle between Y-TZP and CA-Y-TZP (p > 0.05). The histological appearances of the Y-TZP and CA-Y-TZP implants in the tibia (cortical bone) and medial femoral condyle (trabecular bone) after 12 weeks of implantation are shown in Figures 15 and 16, respectively. New bone formation was observed surrounding implant materials. Remodeling and compaction of the bone–implant interface were complete for both tibial and femoral implants, No signs of an inflammatory response were detected in the tissue surrounding the implants. Tighter BIC was observed for CA-YTZP implants not only tibial but also femoral implantation. The presence of fibrous tissue [Figure 15(a)] and bone marrow [Figure 16(a)] was identified for Y-TZP implants. Newly formed interfacial bone for the tibial implant had a lamellar appearance and more interfacial bone contact was observed with CA-Y-TZP than with Y-TZP. New bone had completely remodeled into mature trabecular bone for the femoral implant. Bone marrow tissue was present between the areas of bone contact. All implant surfaces were partially covered with new bone. CA-Y-TZP showed more bone contact than that of Y-TZP.
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In this study, the biocompatibility of a thin CA-coated Y-TZP, using the molecular precursor method, was evaluated by in vitro SBF immersion experiments and in vivo animal implantation experiments. Cylindrical implants were inserted into the tibiae and femoral condyles of rabbits. Several methods have been proposed for apatite coating. A very thin apatite coating technique such as magnetron sputtering was previously developed to overcome the disadvantages associated with the plasma spray method.29,31 The molecular precursor method is a new wet process for coating thin CA layers onto titanium. The advantage of the molecular precursor method is its simplicity and also that the CA coating can be deposited onto substrates of any shape. The inside of a three-dimensional titanium fiber mesh was previously shown to be coated with a thin CA film using the molecular precursor method.32,33 The interface between the deposited CA film and Y-TZP was observed using a FIB processing technique, which can produce nano-scale specimens without any damage. Tanaka27 reported the structures of two types of titanium oxide interfaces using the FIB technique. A cross-sectional view of CA-YTZP revealed a uniform and firmly adhered CA film on Y-TZP with a thickness of less than 1 mm. A difference in CA film thickness was observed between the inner side and outer side of the disk. The spin coat technique was used in this study to achieve a thin CA coating with the molecular precursor method. We speculated that easy diffusion of the precursor solution toward the outer direction of the disk produced a difference in the thickness of the CA film. XRD and FT-IR measurements were used to characterize CA films, which was consistent with a previous report.24 EDX measurements also identified the Ca and P atoms of the CA coating on the surface of CA-Y-TZP, indicating the uniform coating of thin CA layer.
FIGURE 11. FT-IR spectrum of deposited crystals on Y-TZP and CA-YTZP disks after 7 and 14 days of immersion in HBSS.
BONE RESPONSES TO ZIRCONIA IMPLANTS WITH A THIN CA COATING
FIGURE 12. EPMA analysis of CA film on Y-TZP cylinder. Zr: zirconium, Ca: calcium, and P: phosphorus.
FIGURE 13. CLSM pictures of Y-TZP and CA-Y-TZP cylinders in the tibia (cortical bone) in which fluorochrome labeling was performed with calcein 11 weeks postsurgery. (a) Y-TZP and (b) CA-Y-TZP.
FIGURE 14. CLSM pictures of Y-TZP and CA-Y-TZP cylinders in femoral condyle (trabecular bone) in which fluorochrome labeling was performed with calcein 11 weeks postsurgery. (a) Y-TZP and (b) CA-Y-TZP.
In the SBF immersion experiments, apatite deposition on the CA-Y-TZP disks had progressed more than that on the YTZP disks. To evaluate in vitro biocompatibility, many studies have reported the formation of an apatite layer after materials were immersed in SBF. In this study, HBSS was used as SBF. Hanawa and Ota28 reported that an apatite layer formed on a titanium surface after immersion in HBSS. The in vivo bioactivity (osteoconductivity) of biomaterials such as ceramics was shown to precisely mirror their in vitro apatite-forming ability in SBF,34 namely, the more apatite that formed, the better bone formation was. In vivo bone formation on a CA-coated titanium fiber mesh, reported by Hayakawa et al.,32 confirmed the results of the in vitro SBF immersion experiment. Thus, CA-Y-TZP was expected to enhance bone formation in vivo. In the animal experiments, Y-TZP and CA-Y-TZP cylinders were sand-blasted with large grits and acid etched with HF. This surface treatment was similar to the process known as SLA for titanium implants.35 In consideration of future clinical applications, the above-mentioned surface treatment was applied to cylindrical implants. In this study, uniform CA coating could be deposited on cylindrical shape of Y-TZP by using molecular precursor method. In this study, implants were inserted into cortical tibia bone and trabecular femoral condyle bone. Cortical bone is known to be denser and stiffer than trabecular bone. The elastic modulus of cortical bone was previously shown to be higher than that of trabecular bone.36 The implant site TABLE III. Length of Fluorescence Labeling Length of Fluorescence Labeling (mm) Specimen Y-TZP CA-Y-TZP
Tibia
Femoral Condyle a
10.9 (0.3) 7.9 (0.7)a
Values in brackets are SD. a Significantly different at p < 0.05.
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9.8 (3.1) 6.4 (1.8)
between cortical and trabecular bone has been shown to influence the bone response to implants.37 Hayakawa et al.29 reported that cortical and trabecular bone exhibited different bone responses toward apatite-coated titanium implants. Fluorochrome labeling by calcein demonstrated new bone formation in the surrounding areas of the implants. Fluorescent dyes chelate to calcium ions, resulting in the deposition of a multiple vital label on all actively mineralizing bone surfaces.38 The labeling length for CA-Y-TZP was significant less in the tibia, which corresponded to the lower activity of new bone formation by CA-Y-TZP than by Y-TZP. However, no significant difference was observed in BM around the implants. The maturation of woven bone for CAY-TZP may have occurred at an earlier stage of the bone healing process than that for Y-TZP. The BIC and BM for CA-Y-TZP were significantly higher in the femoral condyle. Our present results for in vivo apatite formation in SBF corresponded with the in vivo bone response. This study revealed that coating CA on Y-TZP using the molecular precursor method promoted new trabecular bone formation surrounding the implants. This was due to improved osteoconductivity by the thin CA coating. The present results indicate that the CA-Y-TZP implant may be suitable for a maxillary site with poor bone quality; however, the length of stability should be confirmed in future studies. No significant difference was observed in BIC and BM between Y-TZP and CA-Y-TZP for cortical bone in the tibia. Differences in the bone response to the Y-TZP implant between cortical bone and trabecular bone may lead to different healing mechanisms, that is, direct bone apposition in tibial cortical bone and endochondral bone formation in the femoral cortical bone. More detailed studies are needed to clarify these different bone responses. Albrektsson et al.39 suggested that osseointegration corresponded to approximately 60% bone contact for titanium
BONE RESPONSES TO ZIRCONIA IMPLANTS WITH A THIN CA COATING
ORIGINAL RESEARCH REPORT
FIGURE 15. Histological appearance of Y-TZP and CA-Y-TZP cylinders in the tibia (cortical bone) after 12 weeks of implantation. (methylene blue and basic fuchsin; original magnification 3100). (a) Y-TZP and (b) CA-Y-TZP.
FIGURE 16. Histological appearance of Y-TZP and CA-Y-TZP cylinders in the femoral condyle (trabecular bone) after 12 weeks of implantation. (methylene blue and basic fuchsin; original magnification 3100). (a) Y-TZP and (b) CA-Y-TZP.
implants. The BIC of the Y-TZP implant in this study was approximately 40–60%. The BIC of the CA-Y-TZP implant for tibial cortical bone was approximately 80%. In contrast, the BIC of the CA-Y-TZP implant for the trabecular femoral condyle was above the limit proposed by Albrestsson et al., but was lower than that of titanium. For example, Hayakawa et al.24 reported that CA-coated titanium implants using the molecular precursor method had a BIC of 70–80% after 12 weeks of implantation into the trabecular bone of rabbit. To improve the bone response, a more detailed study on the thin CA coating, such as the thickness of the CA film or calTABLE IV. Percentage of the Measured BIC and BM BIC (%)
BM (%)
Specimen
Tibia
Femoral Condyle
Tibia
Femoral Condyle
Y-TZP CA-Y-TZP
58.0 (13.8) 79.2 (1.2)
46.1 (4.7)a 67.6 (4.4)a
85.3 (8.8) 96.4 (1.8)
65.2 (10.0)b 79.1 (6.92)b
Values in brackets are SD. a,b Significantly different at p < 0.05.
cium concentration in the molecular precursor solution, is needed. CONCLUSIONS
A thin and uniform CA coating could be deposited on Y-TZP using the molecular precursor method. In the SBF immersion experiment, apatite deposition appeared to have progressed more on the CA-Y-TZP cylinder than on the Y-TZP cylinder. A larger amount of new bone formation was demonstrated in animal experiments and a significantly higher BIC ratio was observed in the trabecular femora condyle. We suggest that thin CA-coated Y-TZP using the molecular precursor method may be applicable as an innovative metal-free implant. ACKNOWLEDGMENTS
We acknowledge Professor Masao Yoshinari, Tokyo Dental College, for his help for the preparation of Y-TZP disk and cylindrical samples and Dr. Kouji Inoue, Tsurumi University School of Dental Medicine, for his help for SEM and EPMA analysis.
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | MONTH 2014 VOL 00B, ISSUE 00
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