- Email: [email protected]
Antimicrobial and osteogenic properties of a hydrophilic-modified nanoscale hydroxyapatite coating on titanium
BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 374 – 382
Research Article
nanomedjournal.com
Antimicrobial and osteogenic properties of a hydrophilic-modified nanoscale hydroxyapatite coating on titanium Asuka Murakami, PhD a , Takafumi Arimoto, PhD b , Dai Suzuki, PhD c , Misato Iwai-Yoshida, PhD d , Fukunaga Otsuka, PhD a , Yo Shibata, PhD a,⁎, Takeshi Igarashi, PhD b , Ryutaro Kamijo, PhD c , Takashi Miyazaki, PhD a a
Department of Oral Biomaterials and Technology, Showa University School of Dentistry, Tokyo, Japan b Department of Oral Microbiology, Showa University School of Dentistry, Tokyo, Japan c Department of Biochemistry, Showa University School of Dentistry, Tokyo, Japan d Department of Orthodontics, Showa University School of Dentistry, Tokyo, Japan Received 30 March 2011; revised 7 June 2011; accepted 11 July 2011
Abstract Hydroxyapatite (HA)-coated titanium (Ti) is commonly used for implantable medical devices. This study examined in vitro osteoblast gene expression and antimicrobial activity against early and late colonizers of supra-gingival plaque on nanoscale HA-coated Ti prepared by discharge in a physiological buffered solution. The HA-coated Ti surface showed super-hydrophilicity, whereas the densely sintered HA and Ti surfaces alone showed lower hydrophilicity. The sintered HA and HA-coated Ti surfaces enhanced osteoblast phenotypes in comparison with the bare Ti surface. The HA-coated Ti enabled antimicrobial activity against early colonizers of supra-gingival plaques, namely Streptococcus mitis and Streptococcus gordonii. Such antimicrobial activity may be caused by the surface hydrophilicity, thereby leading to a repulsion force between the HA-coated Ti surface and the bacterial cell membranes. On the contrary, the sintered HA sample was susceptible to infection of microorganisms. Thus, hydrophilic-modified HA-coated Ti may have potential for use in implantable medical devices. From the Clinical Editor: This study establishes that Hydroxyapatite (HA)-coated titanium (Ti) surface of implanted devices may result in an optimal microenvironment to control and prevent infections and may have potential future clinical applications. © 2012 Elsevier Inc. All rights reserved. Key words: Titanium; Hydroxyapatite Coating; Cell Culture; Osteoblast; Antimicrobial
Hydroxyapatite (HA) coatings have been routinely utilized as bioactive modifications on titanium (Ti) implants, because the coatings enhance bone formation and generally provide early fixation between bone and implant surfaces. 1-6 Despite their widespread use, the relatively low survival rate of HA-coated Ti implants is of particular concern and may be caused by the integrity of bonding between the HA layer and the implant surface. 6-10 The excessive thickness of the HA layer is readily removed over time because of the brittle mechanical properties, poor match with the Ti substrate, inhomogeneities and so on, and it has been reported specifically for dental implants (rather than
No conflict of interest was reported by the authors of this article. This work was supported by a Grant-in-Aid for Scientific Research (B) and a Grant-in-Aid for the Encouragement of Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. ⁎Corresponding author: Department of Oral Biomaterials and Technology, Showa University School of Dentistry, 1-5-8 Hatanodai, Tokyo 142-8555, Japan. E-mail address: [email protected] (Y. Shibata).
orthopedic implants). 11-14 The low survival rate of HA-coated dental implants also indicates that biofilm formation in the oral cavity may be an additional major complication leading to longterm clinical failure. 15-17 Several techniques have been investigated to improve the bonding properties between HA coating layers and Ti substrates concomitant with a reduced thickness of the coating layer. 2,5 , 18-20 However, antimicrobial properties against oral microorganisms have not been applied in the coating techniques. Although several hundred bacterial species are associated with plaque biofilm, mitis-streptococci Streptococcus mitis and Streptococcus gordonii are generally accepted as early colonizers within the supra-gingival plaque biofilm. 21-24 The local accumulation of early colonizer streptococci, followed by late colonizer mutansstreptococci, such as Streptococcus mutans or Streptococcus sobrinus, plays a pivotal role in the maturation of supra-gingival plaque that leads to a potential infection of subgingival plaque. 15 Thus, prevention of early colonizer adherence on HA-coated Ti might delay the infection of a dental implant.
1549-9634/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2011.07.001 Please cite this article as: A., Murakami, et al, Antimicrobial and osteogenic properties of a hydrophilic-modified nanoscale hydroxyapatite coating on titanium. Nanomedicine: NBM 2012;8:374-382, doi:10.1016/j.nano.2011.07.001
A. Murakami et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 374–382
Previous studies have focused on surface chemistry modifications of metallic implants; however, there are still very few antimicrobial modifications of HA-coated Ti implants. 17 , 25-27 A recent approach to preventing bacterial adhesion onto implant surfaces was based on hydrophilicity associated with higher surface energy. 28 This modification leads to reduced bacterial adhesion by increasing the repulsion forces between the modified implant surface and the bacteria, because the surfaces of most bacteria are negatively charged. 15 The HA surface has no electrostatic potential under physiological conditions. 29,30 Thus, an HA surface may not be capable of preventing bacterial adhesion, even though the surface enables early fixation with host bone. Theoretically, pure Ti surfaces exhibit a particular surface energy because of a passive oxide layer. The oxide surface is hydrophilic, binds structural water and forms OH hydrophilic functional groups. 31 The developing thickness of a Ti oxide film therefore results in better surface hydrophilicity associated with higher surface energy. 32,33 Preparation of HA-coated Ti by electrical discharge in a buffered physiological saline solution 9 , 34-37 has several advantages over traditional techniques, such as high temperature plasma spraying. Specifically, it can generate highly crystalline nanoscale HA (approximately 100 nm in diameter) with a thickness of 1 μm. 7,36 The HA crystals are incorporated within the developing thickness of the Ti oxide layer that is generated during processing. In light of these findings, we questioned whether HA-coated Ti prepared by discharge shows antimicrobial activity as well as osteogenic properties. In this study,we first examined the surface energy of HAcoated Ti, sintered HA and a bare Ti plate. The antimicrobial activity induced by the surface hydrophilicity of the samples against early and late colonizers of supra-gingival plaque was determined, as well as the in vitro osteoblast gene expression.
Methods Specimen preparation For experiments, we used JIS grade 2 Ti (Ti 99.7%, N b 0.03%, C b 0.08%, H b 0.013%, Fe b 0.25%, O b 0.20%) (KS-50; Kobe Steel, Tokyo, Japan). Specimens were prepared with dimensions of 10 × 10 × 1.0 mm (Ti). Densely sintered HA specimens (CELLYARD; Pentax Tokyo, Japan) of the same dimensions with a 0% pore ratio were also used. The Ti and HA specimen surfaces were mechanically polished using waterproof polishing papers to #1200 under running water and then finished with alumina particles of 0.3-μm diameter. The prepared specimens were ultrasonically cleaned in acetone, detergent solutions (7X; ICN Biomedicals, Aurora, Ohio) and pure distilled water for 15 minutes. The specimens were dried and stored in a sealed desiccator for 1 week at a humidity of 50% at 23°C. The authors previously demonstrated that nano-calcium phosphate crystals can be prepared by electrically discharging the samples in electrolytes. 9,35,36 The electrolyte solution used for the HA-coated Ti was Hanks' balanced salt solution without organic molecules (HBSS). It was prepared in the laboratory with ion concentrations of 142.0 mmol/L Na +, 5.5 mmol/L K +, 8 mmol/L Mg 2+, 1.26 mmol/L Ca 2+, 140.0 mmol/L Cl –, 8.25
HPO42–
375
HCO3–,
mmol/L and 4.2 mmol/L and buffered to pH 7.4 with adequate HCl. The HBSS was stored in a polypropylene bottle for 1 day at room temperature (23°C) before the subsequent studies were undertaken. The Ti specimen was used as a cathode for the coating device and immersed in HBSS. A platinum plate of 5 × 10 × 0.1 mm was used as a counter electrode. Discharging was generated by increasing the cathodic potential beside the working electrodes in the solutions and between the electrolyte and the working electrode through a gas layer on the surface of the electrode. Discharging was maintained at 1 A and 10 V (416 mA/cm 2) for periods of 540 seconds. The coated specimens (HA-Ti) were washed in distilled water and stored in a desiccator for 24 hours at 23°C in 50% humidity. Surface characterization Characterizations of the sample surfaces were carried out by scanning probe microscopy, transmission electron microscopy (TEM), thin-film x-ray diffraction (TF-XRD), x-ray photoelectron spectroscopy (XPS) and contact angle measurement. The relative thickness of the Ti oxide film was evaluated by XPS. Scanning probe microscopy The nanostructure HA-Ti samples were evaluated by scanning probe microscopy (SPM-9600; Shimadzu, Kyoto, Japan) of a 5 × 5 μm surface area under the contact mode. TEM Conventional TEM (HF-2000; Hitachi, Tokyo, Japan) and bright or dark field imaging were performed on the HA nanoparticles (NPs). Higher magnification imaging was operated at 200 kV. Ti oxide film thickness The Ti and HA-Ti samples were sputtered using an internal ion gun at 10 mA and 0.5 kV (UHV conditions at b 2.66 × 10 –7 mbar). The samples were analyzed at 10 seconds each until 50 seconds. The thickness of each Ti oxide film was evaluated by the relative concentration ratio of oxygen detected to the Ti metal substrate. Contact angle The contact angles of the samples with or without 4 weeks of storage relative to 1 μL of pure distilled water were measured using a contact angle meter (CA-DT; Kyowa Interface Science, Saitama, Japan). The temperature and humidity were kept at 23°C and 50%, respectively, during the measurements. TF-XRD The crystal phases of the sample surfaces were detected by TF-XRD (XRD-6100; Shimadzu) with CuKα radiation. XRD was performed at 40 kV and 40 mA with a scanning speed of 0.02°/4 seconds and a scanning range of 20–50°. XPS The Ti and HA-Ti sample surfaces were analyzed by XPS (ESCA-3400; Shimadzu). High-resolution spectra of Ti2p, O1s,
376
A. Murakami et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 374–382
Figure 1. (A) Scanning probe microscopic observation of an HA-Ti surface with an area of 5 × 5 μm under the contact mode. (B) High-resolution bright and dark-field TEM analyses. The bright and dark field images of the HA NPs indicate that each particle incorporates HA crystals as detected in the diffraction peaks shown in Figure 2, C. Three samples were evaluated in the surface characterization (n = 3). Representative images are shown.
Table 1 The Relative Atomic Concentrations (%) as a function of the sputtering time on Ti and HA-Ti detected by XPS (mean ± SD, n = 3)
Ti 0 sec Ti 10 sec Ti 20 sec Ti 30 sec Ti 40 sec Ti 50 sec HA-Ti 0 sec HA-Ti 10 sec HA-Ti 20 sec HA-Ti 30 sec HA-Ti 40 sec HA-Ti 50 sec
Ti
O
C
Ca
P
Na
23.1 ± 1.4 45.2 ± 1.4 55.5 ± 2.0 59.2 ± 2.2 64.3 ± 2.3 69.6 ± 2.4 3.5 ± 0.2 3.5 ± 0.2 4.8 ± 0.2 5.0 ± 0.3 4.9 ± 0.3 5.0 ± 0.2
53.5 ± 2.5 52.3 ± 1.8 44.5 ± 2.2 40.7 ± 1.5 35.7 ± 1.5 30.3 ± 2.1 65.2 ± 2.6 67.9 ± 2.2 63.1 ± 2.3 67.0 ± 2.5 65.6 ± 1.4 65.4 ± 1.8
23.4 ± 1.2 2.5 ± 0.8 14.0 ± 0.2 1.1 ± 0.1 1.1 ± 0.1 1.4 ± 0.2 1.3 ± 0.1 1.1 ± 0.1
10.1 ± 0.4 16.0 ± 0.3 18.0 ± 0.6 19.0 ± 0.3 18.4 ± 0.2 18.2 ± 0.2
-
1.0 ± 0.1 1.0 ± 0.1
7.2 ± 0.1 11.4 ± 0.2 13.0 ± 0.8 7.6 ± 0.2 8.8 ± 0.2 9.3 ± 0.1
Data were statistically analyzed by ANOVA followed by a post-hoc Tukey test. The Relative Atomic Concentrations (%) as a function of the sputtering time on Ti and HA-Ti detected by XPS (mean ± SD, n = 3). Data were statistically analyzed by ANOVA followed by a post-hoc Tukey test.
C1s, P2p and Na1s were analyzed by MgKα radiation. An emission current of 20 mA and an accelerated voltage of 8 kV were applied in the analysis (UHV conditions at b 5.0 × 10 –14 mbar). The binding energies for each spectrum were calibrated based on the C1s spectra of 285.0 eV. Osteoblastic cell culture Primary calvarial osteoblasts were obtained from the calvariae of neonatal ddY mice (Sankyo Co. Inc., Tokyo, Japan) using 0.1% collagenase and 0.2% dispase. 38 Primary osteoblast cells were cultured in α-MEM (Wako Pure Chemicals Ltd., Osaka, Japan) supplemented with 10% FBS (Invitrogen, Carlsbad, California) and penicillin-streptomycin at 37°C in a CO2 incubator (5% CO2, 95% air). At subconfluency, the cells were detached and seeded onto the samples at a density of 4 ×10 4 cells/cm 2 in culture medium supplemented with 50 μg/mL
ascorbic acid, 10 mM Na-β-glycerophosphate and 10 -8 M dexamethasone. The culture medium was renewed every 3 days. Initial cell adhesion and proliferation assays Cell adhesion property was determined at 3 hours, 3 days and 7 days using a Cell Titer 96 Aqueous One Solution Cell Proliferation Assay with MTS tetrazolium compound [3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] (Promega, Madison, Wisconsin) following the manufacturer's instructions. Quantitative real-time PCR Gene expressions at days 3 and 7 were analyzed by RT-PCR amplification with a SYBR Green method. 33 Total RNA samples were extracted with TRIzol reagent (Invitrogen), and then reverse-transcribed using SuperScript III (Invitrogen).
A. Murakami et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 374–382
377
Quantitative real-time PCR was performed using a SYBR green Fast PCR system (Applied Biosystems, Foster City, California). The primer sequences were as follows: Gapdh, 5′-AAATGGTGAAGGTCGGTGTG-3′ and 5′-TGAAGGGGTCGTTGATGG -3′; Alkaline phosphatase (Alp), 5′-TTCCCACGTTTTCACATTCG-3′ and 5′-GCCAGACCAAAGATGGAGTTG-3′; Type I collagen, 5′-GCCTTGGAGGAAACTTTGCTT-3′ and 5′-GCACGGAAACTCCAGCTGAT-3′; Osteocalcin (Ocn), 5′-CTGACAAAGCCTTCATGTCCAA-3′ and 5′-GGTAGCGCCGGAGTCTGTT-3′; Cyclin D1, 5′-CCTCTCCTGCTACCGCACAA-3′ and 5′-CTAGAGCCGCCAAATTTGCT3′; Runx2, 5′-AGGTTCAACGATCTGAGATTTGTG-3′ and 5′-TGTTATGGTCAAGGTGAAACTCTTG-3′; Osterix, 5′TGGCGTCCTCTCTGCTTGA-3′ and 5′-CTAGAGCCGCCAAATTTGCT-3′; Osteopontin (Opn), 5′-GCTTTTGCCTGTTTGGCATT-3′ and 5′-AGCTGCCAGAATCAGTCACTTTC3′; and Bone sialoprotein 2 (Bsp2), 5′-GAGTTAGCGGCACTCCAACTG-3′ and 5′-CACTTTTGGAGCCCTGCTTT-3′. Each band intensity was quantified and normalized by reference to the Gapdh mRNA expression at 3 and 7 days. ALP activity To examine alkaline phosphatase (ALP) activity histochemically, cells were fixed for 10 minutes with 3.7% formaldehyde at room temperature. After washing with PBS, the cells were incubated for 20 minutes with a mixture of 0.1 mg/mL naphthol AS-MX phosphate (Sigma, St. Louis, Missouri), 0.5% N,Ndimethylformamide, 2 mM MgC12 and 0.6 mg/mL fast blue BB salt (Sigma) in 0.1 M Tris-HC1 (pH 8.5) at room temperature. Representative data for each sample are shown. Determination of antimicrobial efficiency Streptococcus mitis JCM12971, Streptococcus gordonii 10558, Streptococcus mutans 109C and Streptococcus sobrinus ATCC27067 were grown at 37°C under anaerobic conditions at 10% H2, 5% CO2 and 80% N2 for 1 day to the mid-exponential phase in Todd Hewitt broth (Difco Laboratories, Detroit, Michigan). Each bacterial cell suspension was prepared at a density of 4.4 × 10 3 cells/mL. The entire surface of each sample was prepared with 10 μL of bacteria suspension and covered by polypropylene film. The samples were maintained at room temperature for 6 hours and then re-immersed in Todd Hewitt broth. In addition, 10 μL of bacteria suspension without cultivation on the sample surface was immersed in Todd Hewitt broth (control). A 100-times diluted portion of each immersion was spread on a Todd Hewitt agar plate and cultivated under anaerobic conditions at 10% H2, 5% CO2 and 80% N2 for 24 hours. 15,33 The antimicrobial activity was evaluated by the numbers of colonies on the Todd Hewitt agar plates after cultivation.
Figure 2. (A) Relative concentrations of oxygen on Ti and HA-Ti as a function of the sputtering time. (B) Contact angles of pure distilled water on the samples. (C) Representative XRD spectra of samples with a scanning range of 20–50°. The arrows indicate HA peaks on the HA-Ti sample surface. (D) High-resolution XPS spectra of O1s on the samples. The ratios of O 2-, OH - and H2O on Ti are 80.0 ± 2.0%, 18.0 ± 2.0% and 2.0 ± 0.4%, whereas those on HA-Ti are 17.0 ± 1.2%, 56.0 ± 2.0% and 27.0 ± 1.8%, respectively. The relative OH - concentration on HA-Ti is significantly higher (P b 0.01) than that on Ti. Three samples were evaluated in the surface characterization experiments and antimicrobial tests (n = 3). The results are expressed as the mean ± SD (n = 3). Data were statistically analyzed by ANOVA followed by a post-hoc Tukey test. A value of P b 0.01 (⁎) was considered significant.
Statistical analysis Three samples were evaluated in the surface characterization experiments and antimicrobial tests (n = 3), 5 in the cell culture studies (n = 5) and 3 in the quantitative RT-PCR assays (n = 3). The results are expressed as the mean ± SD for each experiment. Data were statistically analyzed by ANOVA followed by a post-hoc Tukey test. A P value of less than 0.01 was considered significant.
Figure 3. In vitro biological activity of the samples. (A) MTS assay on the samples. The asterisks indicate significant differences. (B) Osteoblast gene expressions on the samples at 3 and 7 ds of cell culture. The gene expressions on the samples relative to the Ti surface (defined as a relative expression level of 1) at 3 ds of cell culture are shown. The asterisks indicate significant differences within the same culture stage. Five samples were evaluated in the cell culture studies (n = 5) and 3 in the quantitative RT-PCR assays (n = 3). The results are expressed as the mean ± SD. Data were statistically analyzed by ANOVA followed by a post-hoc Tukey test. A value of P b 0.01 (⁎) was considered significant. (C) Representative images of ALP-positive areas on the sample surfaces. The ALP-positive areas on HA and HA-Ti are greater than that on Ti at 3 ds of culture, whereas the areas are similar at 7 ds of culture.
378
A. Murakami et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 374–382
A. Murakami et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 374–382
379
Results
Numbers of adherent cells on the samples
Surface characterization
According to the absorbance values on the samples, it can be assumed that the numbers of adherent osteoblasts (Figure 3, A) on HA-Ti and HA at 3 hours of cell culture were significantly lower (P b 0.01) than the number on Ti. The numbers of cells on Ti and HA were equivalent (P N 0.01) at 3 days of culture, whereas the number of cells on HA-Ti was still suppressed (P b 0.01) in comparison with the cell numbers on HA and Ti. The number of cells on all samples increased (P b 0.01) at 7 days of culture, with no significant differences (P N 0.01) among the samples.
The HA-Ti surface was fully occupied by a large number of granular nanoscale deposits as observed by scanning probe microscopy (Figure 1, A). There were no specific structures observed on Ti and HA surfaces (not shown). The average roughness of Ti, HA, and HA-Ti were 4.7 ± 0.2 nm, 5.0 ± 0.3 nm, and 48.7 ± 0.7 nm, respectively. The average roughness of HA-Ti was significantly higher (P b 0.01) than those of Ti and HA. High-resolution TEM revealed that HA-Ti was formed by a number of nanoscale crystals (Figure 1, B). The particle size was observed to be around 50 nm. The relative concentration of oxygen on Ti decreased (P b 0.01) with an increase in the sputtering time whereas the concentration of Ti increased (P b 0.01) (Table 1). In contrast, the oxygen concentration on HA-Ti did not decrease (Figure 2, A) and the Ti concentration did not increase (P N 0.01) (Table 1). The higher oxygen concentration over the course of the sputtering time revealed that Ti oxide existed even in the deeper region of the HA-Ti surface. Therefore, developing thickness of the Ti oxide layer on HATi occurred in comparison with Ti. 33 The contact angle of pure distilled water on HA-Ti before storage was b 5 degrees, whereas those on Ti and HA were 38 ± 4 degrees and 43 ± 6 degrees, respectively (Figure 2, B). The contact angle on HA-Ti was significantly lower (P b 0.01) than those on Ti and HA. After 4 weeks of storage in a desiccator, the contact angle of pure distilled water on HA-Ti was 16 ± 1 degree whereas those on Ti and HA were 68 ± 4 degrees and 70 ± 6 degrees, respectively. The contact angle on the samples increased (P b 0.01) over time, but HA-Ti showed a significantly lower contact angle than the other samples, even after the storage period. The 2-theta value of the HA-Ti sample revealed that a hydroxyapatite crystal layer was produced on the Ti surface (Figure 2, C). The high-resolution XPS spectra of O1s on the samples (Figure 2, D) revealed that the main chemical species on Ti and HA-Ti were attributable to O 2-, OH - and H2O. 33 The binding energy values corresponding to these peaks were 530.3, 532.2 and 533.2 eV, respectively. The O2- species on Ti and HA-Ti originated from a superficial Ti oxide film. The ratios of O 2-, OH - and H2O on Ti were 80.0 ± 2.0%, 18.0 ± 2.0% and 2.0 ± 0.4% whereas those on HA-Ti were 17.0 ± 1.2%, 56.0 ± 2.0% and 27.0 ± 1.8%, respectively. The relative OH - concentration on HA-Ti was significantly higher (P b 0.01) than that on Ti. The binding energy values of O1s on HA were 533.4 and 535.1 eV, which were postulated to be PO4 and Ca-OH, respectively. 39 As shown in Figure 2, A, the thickness of a Ti oxide layer was formed on HA-Ti in comparison with Ti. The HA crystals were incorporated within the developing thickness of the Ti oxide layer that was generated during processing. The present high-resolution O1s spectrum on HA-Ti showed a Ti oxide layer rather than HA nanocrystals.
Osteoblast phenotypes on the samples At 3 days of culture, the expressions of Ocn, Osterix, Opn and Bsp2 on HA and HA-Ti were significantly higher (P b 0.01) than those on Ti. The gene expressions in adherent osteoblasts on HA and HA-Ti did not differ significantly (P N 0.01) at 3 days of culture (Figure 3, B). After 7 days of culture (Figure 3, B), the expression of Osterix was still significantly higher (P b 0.01) on HA-Ti than on Ti. The expressions of Opn and Bsp2 on HA and HA-Ti were significantly higher (P b 0.01) than those on Ti, even after 7 days of culture, whereas the expression of Type I collagen on Ti was significantly higher (P b 0.01) than those on HA and HA-Ti. The ALP-positive areas on HA and HA-Ti were greater than that on Ti at 3 days of culture, whereas the areas were similar at 7 days of culture (Figure 3, C). Determination of antimicrobial efficiency The numbers of viable S. mitis and S. gordonii colonies on HA were significantly higher (P b 0.01) than those on HA-Ti and Ti (Figure 4). The numbers of viable S. mitis and S. gordonii on HA-Ti were significantly lower (P b 0.01) than those on HA and Ti. However, the numbers of viable S. mutans and S. sobrinus on the samples were equivalent, with no significant differences (P N 0.01) among the samples.
Discussion HA-Ti showed a developing thickness of Ti oxide film with concomitant growth of hydrophilic functional groups on the surface. Thermally or anodically oxidized Ti surfaces have been reported to show increased surface hydrophilicity because of the formation of OH hydrophilic functional groups 31,33 with the developing thickness of the Ti oxide films. Therefore, the HATi surfaces showed super-hydrophilicity, defined as a contact angle of b 5 degrees, 33 whereas the HA and Ti surfaces exhibited lesser hydrophilicity.
Figure 4. Determination of antimicrobial activity on the samples. Each bacterial cell suspension was prepared at density of 4.4 × 10 3 cells/mL. The entire surface of each sample was prepared with 10 μL of bacteria suspension and covered by polypropylene film. The samples were maintained at room temperature for 6 hrs and then re-immersed in Todd Hewitt broth. In addition, 10 μL of bacteria suspension without cultivation on a sample surface was immersed in Todd Hewitt broth (control). (A) Numbers of viable bacterial cells on the samples after 24 hrs of cultivation. Three samples were evaluated in the antimicrobial tests. The results are expressed as the mean ± SD (n = 3). Data were statistically analyzed by ANOVA followed by a post-hoc Tukey test. A value of P b 0.01 (⁎) was considered significant. (B) Representative pictures of the viable bacterial colonies on Todd Hewitt agar plates after 24 hrs of cultivation.
380
A. Murakami et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 374–382
A. Murakami et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 374–382
Despite its surface super-hydrophilicity, HA-Ti showed a lower cell adhesion property, similar to that observed on HA. The nanoscale HA crystals were incorporated within the developing thickness of the Ti oxide layer on HA-Ti that was generated during processing. A previous study reported that calcium ions were not detectable at the in vitro initial cell adhesion stage on Ti surfaces, whereas the accumulation of sodium or phosphate improved adsorption of noncollagenous serum proteins. 40 Adsorption of noncollagenous serum proteins such as fibronectin or vitronectin on material surfaces plays a critical role in initial cell adhesion to the surfaces. 40 These findings may also imply that local accumulation of calcium ions on the Ti surfaces resisted cell adhesion along with the lesser amount of noncollagenous protein adsorption at the early stage. Because a reduced particle diameter can increase the specific surface area of HA that comes into contact with culture medium or body fluids, the number of cells was distinctively suppressed on HA-Ti rather than on sintered HA. In general, osteoblast differentiation, specializing in mineralization has been evaluated by enhancing the expression of more than 2 types of osteogenic markers in adherent cells to the material surfaces. This was specifically determined by the expression of Ocn, Opn or Bsp 2. Although HA did not show superior initial adhesion properties toward the cells, HA and HATi showed enhanced osteoblast phenotypes in comparison with Ti. Our previous studies indicated that HA specifically conjugates to collagenous matrix proteins rather than noncollagenous serum proteins, such as fibronectin or vitronectin. 35,37 The complex macromolecular assembly of calcium phosphate with extracellular collagenous proteins enables scaffold properties for the adherent osteoblasts. 34,35,37 Thus, it is likely that HA and HA-Ti enhanced the adherent osteoblast phenotypes regardless of the lower cell adhesion properties. Despite the 1-μm thickness of the coating layer, HA-Ti allowed enhanced osteoblast phenotypes, as well as the sintered HA samples, because nanoscale HA can readily conjugate to extracellular collagenous proteins. 41,42 HA-Ti showed antimicrobial activity specifically against early colonizers such as S. mitis and S. gordonii. Only HA-Ti generates an amount of hydrophilic functional groups on the surface because of the thickness of the Ti oxide layer. Therefore, the HA-Ti surface shows a higher negative charge because of these hydrophilic functional groups in comparison with the HA and Ti surfaces. The adhesion mechanisms of the early and late colonizers on the material surfaces have not been fully elucidated. However, because HA-Ti showed antimicrobial activity specifically against S. mitis and S. gordonii, the adhesion mechanisms of these early colonizers may involve an electrostatic force between the material surface and the bacterial cell membranes. On the contrary, the adhesion of the late colonizers S. mutans and S. sobrinus might not involve electrostatic mechanisms. It can be assumed that the antimicrobial activity on HA-Ti was caused by the surface negative charge associated with the OH hydrophilic functional groups, thereby creating a repulsion force between the surface and the bacterial cells. 28 A Ti oxide film formed on Ti surfaces generates hydrophilic functional groups in cell culture conditions, 31 whereas pure HA surfaces exhibit no electrostatic potential. Although the water
381
contact angles on both the HA and bare Ti surfaces did not differ significantly, the Ti surface seemed to generate a repulsion force between the surface and the bacterial membranes, in comparison with the HA surface. The lower electrical charge on the HA surface without hydrophilic functional groups yielded a higher survival rate of adherent early colonizers compared with the HATi and Ti surfaces. Antibiotic-loaded porous HA coatings on Ti have been reported. 17,27 It has been reported that this method leads to burst release of the antibiotics, that is, more than 80–90% of the antibiotic being released from the coating layer within the first 60 minutes. 43,44 Despite the limitation of antimicrobial activity against oral microorganisms, HA-Ti can be expected to ensure longer efficacy rather than antibiotic loading owing to the hydrophilicity. The present findings demonstrated in vitro osteogenic properties, as well as antimicrobial activity, on HA-Ti. HA-Ti has been reported to show in vivo biomechanical integrity because of the micrometer thickness of the functionally graded coating characteristics toward Ti metal substrates. 7,9 Thus, HATi may have potential for use as a surface modification of implantable medical devices, especially dental implants.
References 1. De Groot K, Geesink R, Klein CPAT, Serekian P. Plasma sprayed coatings of hydroxylapatite. J Biomed Mater Res 1987;21:1375-81. 2. Dinda GP, Shin J, Mazumder J. Pulsed laser deposition of hydroxyapatite thin films on Ti-6Al-4V: Effect of heat treatment on structure and properties. Acta Biomater 2009;5:1821-30. 3. Geesink RGT, De Groot K, Klein CPAT. Bonding of bone to apatitecoated implants. J Bone Joint Surg - Series B 1988;70:17-22. 4. Gotfredsen K, Wennerberg A, Johansson C, Skovgaard LT, HjortingHansen E. Anchorage of TiO2-blasted, HA-coated, and machined implants: An experimental study with rabbits. J Biomed Mater Res 1995;29:1223-31. 5. Le Guehennec L, Soueidan A, Layrolle P, Amouriq Y. Surface treatments of titanium dental implants for rapid osseointegration. Dent Mater 2007;23:844-54. 6. Tsui YC, Doyle C, Clyne TW. Plasma sprayed hydroxyapatite coatings on titanium substrates. Part 1: Mechanical properties and residual stress levels. Biomaterials 1998;19:2015-29. 7. Shibata Y, Takashima H, Yamamoto H, Miyazaki T. Functionally gradient bonelike hydroxyapatite coating on a titanium metal substrate created by a discharging method in HBSS without organic molecules. Int J Oral Maxillofac Implants 2004;19:177-83. 8. Wheeler SL. Eight-year clinical retrospective study of titanium plasmasprayed and hydroxyapatite-coated cylinder implants. Int J Oral Maxillofac Implants 1996;11:340-50. 9. Yamamoto H, Shibata Y, Tachikawa T, Miyazaki T. In vivo performance of two different hydroxyapatite coatings on titanium prepared by discharging in electrolytes. J Biomed Mater Res - Part B Applied Biomaterials 2006;78:211-4. 10. Zheng X, Huang M, Ding C. Bond strength of plasma-sprayed hydroxyapatite/Ti composite coatings. Biomaterials 2000;21:841-9. 11. Albrektsson T, Sennerby L, Wennerberg A. State of the art of oral implants. Periodontology 2000 2008;47:15-26. 12. Chang JK, Chen CH, Huang KY, Wang GJ. Eight-Year Results of Hydroxyapatite-Coated Hip Arthroplasty. J Arthroplasty 2006;21:541-6. 13. Geesink RGT, Hoefnagels NHM. Six-year results of hydroxyapatitecoated total hip replacement. J Bone Joint Surg - Series B 1995;77: 534-47.
382
A. Murakami et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 374–382
14. Callan DP, Cobb CM, Williams KBDNA. probe identification of bacteria colonizing internal surfaces of the implant-abutment interface: A preliminary study. J Periodontol 2005;76:115-20. 15. Omori S, Shibata Y, Arimoto T, Igarashi T, Baba K, Miyazaki T. Microorganism and cell viability on antimicrobially modified titanium. J Dent Res 2009;88:957-62. 16. Deng JY, Arimoto T, Shibata Y, Omori S, Miyazaki T, Igarashi T. Role of chloride formed on anodized titanium surfaces against an oral microorganism. J Biomater Appl 2010;25:179-89. 17. Zhao L, Chu PK, Zhang Y, Wu Z. Antibacterial coatings on titanium implants. J Biomed Mater Res-Part B Appl Biomater 2009;91:470-80. 18. Gao Y, Zou S, Liu X, Bao C, Hu J. The effect of surface immobilized bisphosphonates on the fixation of hydroxyapatite-coated titanium implants in ovariectomized rats. Biomaterials 2009;30:1790-6. 19. O'Hare P, Meenan BJ, Burke GA, Byrne G, Dowling D, Hunt JA. Biological responses to hydroxyapatite surfaces deposited via a coincident microblasting technique. Biomaterials 2010;31:515-22. 20. Saber-Samandari S, Gross KA. The use of thermal printing to control the properties of calcium phosphate deposits. Biomaterials 2010;31:6386-93. 21. Slots J, Listgarten MA. Bacteroides gingivalis, bacteroides intermedius and actinobacillus actinomycetemcomitans in human periodontal diseases. J Clin Periodontol 1988;15:85-93. 22. Moore WE. Microbiology of periodontal disease. J Periodont Res 1987;22:335-41. 23. Medaglini D, Pozzi G, King TP, Fischetti VA. Mucosal and systemic immune responses to a recombinant protein expressed on the surface of the oral commensal bacterium Streptococcus gordonii after oral colonization. Proc Nat Acad Sci U S A 1995;92:6868-72. 24. Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL. Microbial complexes in subgingival plaque. J Clin Periodontol 1998;25:134-44. 25. Ge X, Leng Y, Bao C, Xu SL, Wang R, Ren F. Antibacterial coatings of fluoridated hydroxyapatite for percutaneous implants. J Biomed Mater Res-Part A 2010;95 A:588-99. 26. Mo A, Liao J, Xu W, Xian S, Li Y, Bai S. Preparation and antibacterial effect of silver-hydroxyapatite/titania nanocomposite thin film on titanium. Appl Surf Sci 2008;255:435-8. 27. Stigter M, Bezemer J, De Groot K, Layrolle P. Incorporation of different antibiotics into carbonated hydroxyapatite coatings on titanium implants, release and antibiotic efficacy. J Control Release 2004;99:127-37. 28. Chua PH, Neoh KG, Kang ET, Wang W. Surface functionalization of titanium with hyaluronic acid/chitosan polyelectrolyte multilayers and RGD for promoting osteoblast functions and inhibiting bacterial adhesion. Biomaterials 2008;29:1412-21. 29. Zhitomirsky I, Gal-Or L. Electrophoretic deposition of hydroxyapatite. J Mater Sci: Mater Med 1997;8:213-9.
30. Ma J, Wang C, Peng KW. Electrophoretic deposition of porous hydroxyapatite scaffold. Biomaterials 2003;24:3505-10. 31. Zhao G, Schwartz Z, Wieland M, Rupp F, Geis-Gerstorfer J, Cochran DL, et al. High surface energy enhances cell response to titanium substrate microstructure. J Biomedl Mater Res - Part A 2005;74:49-58. 32. Park YJ, Song HJ, Kim I, Yang HS. Surface characteristics and bioactivity of oxide film on titanium metal formed by thermal oxidation. J Mater Sci Mater Med 2007;18:565-75. 33. Shibata Y, Suzuki D, Omori S, Tanaka R, Murakami A, Kataoka Y, et al. The characteristics of in vitro biological activity of titanium surfaces anodically oxidized in chloride solutions. Biomaterials 2010;31: 8546-55. 34. Shibata Y, He LH, Toda Y, Kataoka Y, Fujisawa N, Miyazaki T, et al. Micromechanical evaluation of mineralized multilayers. J Biomech 2008;41:3414-8. 35. Toda Y, Shibata Y, Kataoka Y, Kawawa T, Miyazaki T. Interfacial assembly of bioinspired nanostructures mediated by supersensitive crystals. J Biomed Mater Res-Part A 2008;84:869-74. 36. Takashima H, Shibata Y, Kim TY, Miyazaki T. Hydroxyapatite coating on a titanium metal substrate by a discharging method in modified artificial body fluid. Int J Oral Maxillofac Implants 2004;19: 66-72. 37. Shibata Y, Yamamoto H, Miyazaki T. Colloidal beta-tricalcium phosphate prepared by discharge in a modified body fluid facilitates synthesis of collagen composites. J Dent Res 2005;84:827-31. 38. Suda T, Jimi E, Nakamura I, Takahashi N. Role of 1 alpha,25dihydroxyvitamin D3 in osteoclast differentiation and function. Methods Enzymol 1997;283:223-35. 39. Santos JD, Jha LJ, Monteiro FJ. In vitro calcium phosphate formation on SiO2-Na2O-CaO-P2O5 glass reinforced hydroxyapatite composite: A study by XPS analysis. J Mater Sci Mater Med 1996;7:181-5. 40. Yamamoto H, Shibata Y, Miyazaki T. Anode glow discharge plasma treatment of titanium plates facilitates adsorption of extracellular matrix proteins to the plates. J Dent Res 2005;84:668-71. 41. Chang MC, Tanaka J. FT-IR study for hydroxyapatite/collagen nanocomposite cross-linked by glutaraldehyde. Biomaterials 2002;23: 4811-8. 42. Chang MC, Tanaka J. XPS study for the microstructure development of hydroxyapatite-collagen nanocomposites cross-linked using glutaraldehyde. Biomaterials 2002;23:3879-85. 43. Radin S, Campbell JT, Ducheyne P, Cuckler JM. Calcium phosphate ceramic coatings as carriers of vancomycin. Biomaterials 1997;18:777-82. 44. Yamamura K, Iwata H, Yotsuyanagi T. Synthesis of antibiotic-loaded hydroxyapatite beads and in vitro drug release testing. J Biomed Mater Res 1992;26:1053-64.
Research Article
nanomedjournal.com
Antimicrobial and osteogenic properties of a hydrophilic-modified nanoscale hydroxyapatite coating on titanium Asuka Murakami, PhD a , Takafumi Arimoto, PhD b , Dai Suzuki, PhD c , Misato Iwai-Yoshida, PhD d , Fukunaga Otsuka, PhD a , Yo Shibata, PhD a,⁎, Takeshi Igarashi, PhD b , Ryutaro Kamijo, PhD c , Takashi Miyazaki, PhD a a
Department of Oral Biomaterials and Technology, Showa University School of Dentistry, Tokyo, Japan b Department of Oral Microbiology, Showa University School of Dentistry, Tokyo, Japan c Department of Biochemistry, Showa University School of Dentistry, Tokyo, Japan d Department of Orthodontics, Showa University School of Dentistry, Tokyo, Japan Received 30 March 2011; revised 7 June 2011; accepted 11 July 2011
Abstract Hydroxyapatite (HA)-coated titanium (Ti) is commonly used for implantable medical devices. This study examined in vitro osteoblast gene expression and antimicrobial activity against early and late colonizers of supra-gingival plaque on nanoscale HA-coated Ti prepared by discharge in a physiological buffered solution. The HA-coated Ti surface showed super-hydrophilicity, whereas the densely sintered HA and Ti surfaces alone showed lower hydrophilicity. The sintered HA and HA-coated Ti surfaces enhanced osteoblast phenotypes in comparison with the bare Ti surface. The HA-coated Ti enabled antimicrobial activity against early colonizers of supra-gingival plaques, namely Streptococcus mitis and Streptococcus gordonii. Such antimicrobial activity may be caused by the surface hydrophilicity, thereby leading to a repulsion force between the HA-coated Ti surface and the bacterial cell membranes. On the contrary, the sintered HA sample was susceptible to infection of microorganisms. Thus, hydrophilic-modified HA-coated Ti may have potential for use in implantable medical devices. From the Clinical Editor: This study establishes that Hydroxyapatite (HA)-coated titanium (Ti) surface of implanted devices may result in an optimal microenvironment to control and prevent infections and may have potential future clinical applications. © 2012 Elsevier Inc. All rights reserved. Key words: Titanium; Hydroxyapatite Coating; Cell Culture; Osteoblast; Antimicrobial
Hydroxyapatite (HA) coatings have been routinely utilized as bioactive modifications on titanium (Ti) implants, because the coatings enhance bone formation and generally provide early fixation between bone and implant surfaces. 1-6 Despite their widespread use, the relatively low survival rate of HA-coated Ti implants is of particular concern and may be caused by the integrity of bonding between the HA layer and the implant surface. 6-10 The excessive thickness of the HA layer is readily removed over time because of the brittle mechanical properties, poor match with the Ti substrate, inhomogeneities and so on, and it has been reported specifically for dental implants (rather than
No conflict of interest was reported by the authors of this article. This work was supported by a Grant-in-Aid for Scientific Research (B) and a Grant-in-Aid for the Encouragement of Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. ⁎Corresponding author: Department of Oral Biomaterials and Technology, Showa University School of Dentistry, 1-5-8 Hatanodai, Tokyo 142-8555, Japan. E-mail address: [email protected] (Y. Shibata).
orthopedic implants). 11-14 The low survival rate of HA-coated dental implants also indicates that biofilm formation in the oral cavity may be an additional major complication leading to longterm clinical failure. 15-17 Several techniques have been investigated to improve the bonding properties between HA coating layers and Ti substrates concomitant with a reduced thickness of the coating layer. 2,5 , 18-20 However, antimicrobial properties against oral microorganisms have not been applied in the coating techniques. Although several hundred bacterial species are associated with plaque biofilm, mitis-streptococci Streptococcus mitis and Streptococcus gordonii are generally accepted as early colonizers within the supra-gingival plaque biofilm. 21-24 The local accumulation of early colonizer streptococci, followed by late colonizer mutansstreptococci, such as Streptococcus mutans or Streptococcus sobrinus, plays a pivotal role in the maturation of supra-gingival plaque that leads to a potential infection of subgingival plaque. 15 Thus, prevention of early colonizer adherence on HA-coated Ti might delay the infection of a dental implant.
1549-9634/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2011.07.001 Please cite this article as: A., Murakami, et al, Antimicrobial and osteogenic properties of a hydrophilic-modified nanoscale hydroxyapatite coating on titanium. Nanomedicine: NBM 2012;8:374-382, doi:10.1016/j.nano.2011.07.001
A. Murakami et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 374–382
Previous studies have focused on surface chemistry modifications of metallic implants; however, there are still very few antimicrobial modifications of HA-coated Ti implants. 17 , 25-27 A recent approach to preventing bacterial adhesion onto implant surfaces was based on hydrophilicity associated with higher surface energy. 28 This modification leads to reduced bacterial adhesion by increasing the repulsion forces between the modified implant surface and the bacteria, because the surfaces of most bacteria are negatively charged. 15 The HA surface has no electrostatic potential under physiological conditions. 29,30 Thus, an HA surface may not be capable of preventing bacterial adhesion, even though the surface enables early fixation with host bone. Theoretically, pure Ti surfaces exhibit a particular surface energy because of a passive oxide layer. The oxide surface is hydrophilic, binds structural water and forms OH hydrophilic functional groups. 31 The developing thickness of a Ti oxide film therefore results in better surface hydrophilicity associated with higher surface energy. 32,33 Preparation of HA-coated Ti by electrical discharge in a buffered physiological saline solution 9 , 34-37 has several advantages over traditional techniques, such as high temperature plasma spraying. Specifically, it can generate highly crystalline nanoscale HA (approximately 100 nm in diameter) with a thickness of 1 μm. 7,36 The HA crystals are incorporated within the developing thickness of the Ti oxide layer that is generated during processing. In light of these findings, we questioned whether HA-coated Ti prepared by discharge shows antimicrobial activity as well as osteogenic properties. In this study,we first examined the surface energy of HAcoated Ti, sintered HA and a bare Ti plate. The antimicrobial activity induced by the surface hydrophilicity of the samples against early and late colonizers of supra-gingival plaque was determined, as well as the in vitro osteoblast gene expression.
Methods Specimen preparation For experiments, we used JIS grade 2 Ti (Ti 99.7%, N b 0.03%, C b 0.08%, H b 0.013%, Fe b 0.25%, O b 0.20%) (KS-50; Kobe Steel, Tokyo, Japan). Specimens were prepared with dimensions of 10 × 10 × 1.0 mm (Ti). Densely sintered HA specimens (CELLYARD; Pentax Tokyo, Japan) of the same dimensions with a 0% pore ratio were also used. The Ti and HA specimen surfaces were mechanically polished using waterproof polishing papers to #1200 under running water and then finished with alumina particles of 0.3-μm diameter. The prepared specimens were ultrasonically cleaned in acetone, detergent solutions (7X; ICN Biomedicals, Aurora, Ohio) and pure distilled water for 15 minutes. The specimens were dried and stored in a sealed desiccator for 1 week at a humidity of 50% at 23°C. The authors previously demonstrated that nano-calcium phosphate crystals can be prepared by electrically discharging the samples in electrolytes. 9,35,36 The electrolyte solution used for the HA-coated Ti was Hanks' balanced salt solution without organic molecules (HBSS). It was prepared in the laboratory with ion concentrations of 142.0 mmol/L Na +, 5.5 mmol/L K +, 8 mmol/L Mg 2+, 1.26 mmol/L Ca 2+, 140.0 mmol/L Cl –, 8.25
HPO42–
375
HCO3–,
mmol/L and 4.2 mmol/L and buffered to pH 7.4 with adequate HCl. The HBSS was stored in a polypropylene bottle for 1 day at room temperature (23°C) before the subsequent studies were undertaken. The Ti specimen was used as a cathode for the coating device and immersed in HBSS. A platinum plate of 5 × 10 × 0.1 mm was used as a counter electrode. Discharging was generated by increasing the cathodic potential beside the working electrodes in the solutions and between the electrolyte and the working electrode through a gas layer on the surface of the electrode. Discharging was maintained at 1 A and 10 V (416 mA/cm 2) for periods of 540 seconds. The coated specimens (HA-Ti) were washed in distilled water and stored in a desiccator for 24 hours at 23°C in 50% humidity. Surface characterization Characterizations of the sample surfaces were carried out by scanning probe microscopy, transmission electron microscopy (TEM), thin-film x-ray diffraction (TF-XRD), x-ray photoelectron spectroscopy (XPS) and contact angle measurement. The relative thickness of the Ti oxide film was evaluated by XPS. Scanning probe microscopy The nanostructure HA-Ti samples were evaluated by scanning probe microscopy (SPM-9600; Shimadzu, Kyoto, Japan) of a 5 × 5 μm surface area under the contact mode. TEM Conventional TEM (HF-2000; Hitachi, Tokyo, Japan) and bright or dark field imaging were performed on the HA nanoparticles (NPs). Higher magnification imaging was operated at 200 kV. Ti oxide film thickness The Ti and HA-Ti samples were sputtered using an internal ion gun at 10 mA and 0.5 kV (UHV conditions at b 2.66 × 10 –7 mbar). The samples were analyzed at 10 seconds each until 50 seconds. The thickness of each Ti oxide film was evaluated by the relative concentration ratio of oxygen detected to the Ti metal substrate. Contact angle The contact angles of the samples with or without 4 weeks of storage relative to 1 μL of pure distilled water were measured using a contact angle meter (CA-DT; Kyowa Interface Science, Saitama, Japan). The temperature and humidity were kept at 23°C and 50%, respectively, during the measurements. TF-XRD The crystal phases of the sample surfaces were detected by TF-XRD (XRD-6100; Shimadzu) with CuKα radiation. XRD was performed at 40 kV and 40 mA with a scanning speed of 0.02°/4 seconds and a scanning range of 20–50°. XPS The Ti and HA-Ti sample surfaces were analyzed by XPS (ESCA-3400; Shimadzu). High-resolution spectra of Ti2p, O1s,
376
A. Murakami et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 374–382
Figure 1. (A) Scanning probe microscopic observation of an HA-Ti surface with an area of 5 × 5 μm under the contact mode. (B) High-resolution bright and dark-field TEM analyses. The bright and dark field images of the HA NPs indicate that each particle incorporates HA crystals as detected in the diffraction peaks shown in Figure 2, C. Three samples were evaluated in the surface characterization (n = 3). Representative images are shown.
Table 1 The Relative Atomic Concentrations (%) as a function of the sputtering time on Ti and HA-Ti detected by XPS (mean ± SD, n = 3)
Ti 0 sec Ti 10 sec Ti 20 sec Ti 30 sec Ti 40 sec Ti 50 sec HA-Ti 0 sec HA-Ti 10 sec HA-Ti 20 sec HA-Ti 30 sec HA-Ti 40 sec HA-Ti 50 sec
Ti
O
C
Ca
P
Na
23.1 ± 1.4 45.2 ± 1.4 55.5 ± 2.0 59.2 ± 2.2 64.3 ± 2.3 69.6 ± 2.4 3.5 ± 0.2 3.5 ± 0.2 4.8 ± 0.2 5.0 ± 0.3 4.9 ± 0.3 5.0 ± 0.2
53.5 ± 2.5 52.3 ± 1.8 44.5 ± 2.2 40.7 ± 1.5 35.7 ± 1.5 30.3 ± 2.1 65.2 ± 2.6 67.9 ± 2.2 63.1 ± 2.3 67.0 ± 2.5 65.6 ± 1.4 65.4 ± 1.8
23.4 ± 1.2 2.5 ± 0.8 14.0 ± 0.2 1.1 ± 0.1 1.1 ± 0.1 1.4 ± 0.2 1.3 ± 0.1 1.1 ± 0.1
10.1 ± 0.4 16.0 ± 0.3 18.0 ± 0.6 19.0 ± 0.3 18.4 ± 0.2 18.2 ± 0.2
-
1.0 ± 0.1 1.0 ± 0.1
7.2 ± 0.1 11.4 ± 0.2 13.0 ± 0.8 7.6 ± 0.2 8.8 ± 0.2 9.3 ± 0.1
Data were statistically analyzed by ANOVA followed by a post-hoc Tukey test. The Relative Atomic Concentrations (%) as a function of the sputtering time on Ti and HA-Ti detected by XPS (mean ± SD, n = 3). Data were statistically analyzed by ANOVA followed by a post-hoc Tukey test.
C1s, P2p and Na1s were analyzed by MgKα radiation. An emission current of 20 mA and an accelerated voltage of 8 kV were applied in the analysis (UHV conditions at b 5.0 × 10 –14 mbar). The binding energies for each spectrum were calibrated based on the C1s spectra of 285.0 eV. Osteoblastic cell culture Primary calvarial osteoblasts were obtained from the calvariae of neonatal ddY mice (Sankyo Co. Inc., Tokyo, Japan) using 0.1% collagenase and 0.2% dispase. 38 Primary osteoblast cells were cultured in α-MEM (Wako Pure Chemicals Ltd., Osaka, Japan) supplemented with 10% FBS (Invitrogen, Carlsbad, California) and penicillin-streptomycin at 37°C in a CO2 incubator (5% CO2, 95% air). At subconfluency, the cells were detached and seeded onto the samples at a density of 4 ×10 4 cells/cm 2 in culture medium supplemented with 50 μg/mL
ascorbic acid, 10 mM Na-β-glycerophosphate and 10 -8 M dexamethasone. The culture medium was renewed every 3 days. Initial cell adhesion and proliferation assays Cell adhesion property was determined at 3 hours, 3 days and 7 days using a Cell Titer 96 Aqueous One Solution Cell Proliferation Assay with MTS tetrazolium compound [3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] (Promega, Madison, Wisconsin) following the manufacturer's instructions. Quantitative real-time PCR Gene expressions at days 3 and 7 were analyzed by RT-PCR amplification with a SYBR Green method. 33 Total RNA samples were extracted with TRIzol reagent (Invitrogen), and then reverse-transcribed using SuperScript III (Invitrogen).
A. Murakami et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 374–382
377
Quantitative real-time PCR was performed using a SYBR green Fast PCR system (Applied Biosystems, Foster City, California). The primer sequences were as follows: Gapdh, 5′-AAATGGTGAAGGTCGGTGTG-3′ and 5′-TGAAGGGGTCGTTGATGG -3′; Alkaline phosphatase (Alp), 5′-TTCCCACGTTTTCACATTCG-3′ and 5′-GCCAGACCAAAGATGGAGTTG-3′; Type I collagen, 5′-GCCTTGGAGGAAACTTTGCTT-3′ and 5′-GCACGGAAACTCCAGCTGAT-3′; Osteocalcin (Ocn), 5′-CTGACAAAGCCTTCATGTCCAA-3′ and 5′-GGTAGCGCCGGAGTCTGTT-3′; Cyclin D1, 5′-CCTCTCCTGCTACCGCACAA-3′ and 5′-CTAGAGCCGCCAAATTTGCT3′; Runx2, 5′-AGGTTCAACGATCTGAGATTTGTG-3′ and 5′-TGTTATGGTCAAGGTGAAACTCTTG-3′; Osterix, 5′TGGCGTCCTCTCTGCTTGA-3′ and 5′-CTAGAGCCGCCAAATTTGCT-3′; Osteopontin (Opn), 5′-GCTTTTGCCTGTTTGGCATT-3′ and 5′-AGCTGCCAGAATCAGTCACTTTC3′; and Bone sialoprotein 2 (Bsp2), 5′-GAGTTAGCGGCACTCCAACTG-3′ and 5′-CACTTTTGGAGCCCTGCTTT-3′. Each band intensity was quantified and normalized by reference to the Gapdh mRNA expression at 3 and 7 days. ALP activity To examine alkaline phosphatase (ALP) activity histochemically, cells were fixed for 10 minutes with 3.7% formaldehyde at room temperature. After washing with PBS, the cells were incubated for 20 minutes with a mixture of 0.1 mg/mL naphthol AS-MX phosphate (Sigma, St. Louis, Missouri), 0.5% N,Ndimethylformamide, 2 mM MgC12 and 0.6 mg/mL fast blue BB salt (Sigma) in 0.1 M Tris-HC1 (pH 8.5) at room temperature. Representative data for each sample are shown. Determination of antimicrobial efficiency Streptococcus mitis JCM12971, Streptococcus gordonii 10558, Streptococcus mutans 109C and Streptococcus sobrinus ATCC27067 were grown at 37°C under anaerobic conditions at 10% H2, 5% CO2 and 80% N2 for 1 day to the mid-exponential phase in Todd Hewitt broth (Difco Laboratories, Detroit, Michigan). Each bacterial cell suspension was prepared at a density of 4.4 × 10 3 cells/mL. The entire surface of each sample was prepared with 10 μL of bacteria suspension and covered by polypropylene film. The samples were maintained at room temperature for 6 hours and then re-immersed in Todd Hewitt broth. In addition, 10 μL of bacteria suspension without cultivation on the sample surface was immersed in Todd Hewitt broth (control). A 100-times diluted portion of each immersion was spread on a Todd Hewitt agar plate and cultivated under anaerobic conditions at 10% H2, 5% CO2 and 80% N2 for 24 hours. 15,33 The antimicrobial activity was evaluated by the numbers of colonies on the Todd Hewitt agar plates after cultivation.
Figure 2. (A) Relative concentrations of oxygen on Ti and HA-Ti as a function of the sputtering time. (B) Contact angles of pure distilled water on the samples. (C) Representative XRD spectra of samples with a scanning range of 20–50°. The arrows indicate HA peaks on the HA-Ti sample surface. (D) High-resolution XPS spectra of O1s on the samples. The ratios of O 2-, OH - and H2O on Ti are 80.0 ± 2.0%, 18.0 ± 2.0% and 2.0 ± 0.4%, whereas those on HA-Ti are 17.0 ± 1.2%, 56.0 ± 2.0% and 27.0 ± 1.8%, respectively. The relative OH - concentration on HA-Ti is significantly higher (P b 0.01) than that on Ti. Three samples were evaluated in the surface characterization experiments and antimicrobial tests (n = 3). The results are expressed as the mean ± SD (n = 3). Data were statistically analyzed by ANOVA followed by a post-hoc Tukey test. A value of P b 0.01 (⁎) was considered significant.
Statistical analysis Three samples were evaluated in the surface characterization experiments and antimicrobial tests (n = 3), 5 in the cell culture studies (n = 5) and 3 in the quantitative RT-PCR assays (n = 3). The results are expressed as the mean ± SD for each experiment. Data were statistically analyzed by ANOVA followed by a post-hoc Tukey test. A P value of less than 0.01 was considered significant.
Figure 3. In vitro biological activity of the samples. (A) MTS assay on the samples. The asterisks indicate significant differences. (B) Osteoblast gene expressions on the samples at 3 and 7 ds of cell culture. The gene expressions on the samples relative to the Ti surface (defined as a relative expression level of 1) at 3 ds of cell culture are shown. The asterisks indicate significant differences within the same culture stage. Five samples were evaluated in the cell culture studies (n = 5) and 3 in the quantitative RT-PCR assays (n = 3). The results are expressed as the mean ± SD. Data were statistically analyzed by ANOVA followed by a post-hoc Tukey test. A value of P b 0.01 (⁎) was considered significant. (C) Representative images of ALP-positive areas on the sample surfaces. The ALP-positive areas on HA and HA-Ti are greater than that on Ti at 3 ds of culture, whereas the areas are similar at 7 ds of culture.
378
A. Murakami et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 374–382
A. Murakami et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 374–382
379
Results
Numbers of adherent cells on the samples
Surface characterization
According to the absorbance values on the samples, it can be assumed that the numbers of adherent osteoblasts (Figure 3, A) on HA-Ti and HA at 3 hours of cell culture were significantly lower (P b 0.01) than the number on Ti. The numbers of cells on Ti and HA were equivalent (P N 0.01) at 3 days of culture, whereas the number of cells on HA-Ti was still suppressed (P b 0.01) in comparison with the cell numbers on HA and Ti. The number of cells on all samples increased (P b 0.01) at 7 days of culture, with no significant differences (P N 0.01) among the samples.
The HA-Ti surface was fully occupied by a large number of granular nanoscale deposits as observed by scanning probe microscopy (Figure 1, A). There were no specific structures observed on Ti and HA surfaces (not shown). The average roughness of Ti, HA, and HA-Ti were 4.7 ± 0.2 nm, 5.0 ± 0.3 nm, and 48.7 ± 0.7 nm, respectively. The average roughness of HA-Ti was significantly higher (P b 0.01) than those of Ti and HA. High-resolution TEM revealed that HA-Ti was formed by a number of nanoscale crystals (Figure 1, B). The particle size was observed to be around 50 nm. The relative concentration of oxygen on Ti decreased (P b 0.01) with an increase in the sputtering time whereas the concentration of Ti increased (P b 0.01) (Table 1). In contrast, the oxygen concentration on HA-Ti did not decrease (Figure 2, A) and the Ti concentration did not increase (P N 0.01) (Table 1). The higher oxygen concentration over the course of the sputtering time revealed that Ti oxide existed even in the deeper region of the HA-Ti surface. Therefore, developing thickness of the Ti oxide layer on HATi occurred in comparison with Ti. 33 The contact angle of pure distilled water on HA-Ti before storage was b 5 degrees, whereas those on Ti and HA were 38 ± 4 degrees and 43 ± 6 degrees, respectively (Figure 2, B). The contact angle on HA-Ti was significantly lower (P b 0.01) than those on Ti and HA. After 4 weeks of storage in a desiccator, the contact angle of pure distilled water on HA-Ti was 16 ± 1 degree whereas those on Ti and HA were 68 ± 4 degrees and 70 ± 6 degrees, respectively. The contact angle on the samples increased (P b 0.01) over time, but HA-Ti showed a significantly lower contact angle than the other samples, even after the storage period. The 2-theta value of the HA-Ti sample revealed that a hydroxyapatite crystal layer was produced on the Ti surface (Figure 2, C). The high-resolution XPS spectra of O1s on the samples (Figure 2, D) revealed that the main chemical species on Ti and HA-Ti were attributable to O 2-, OH - and H2O. 33 The binding energy values corresponding to these peaks were 530.3, 532.2 and 533.2 eV, respectively. The O2- species on Ti and HA-Ti originated from a superficial Ti oxide film. The ratios of O 2-, OH - and H2O on Ti were 80.0 ± 2.0%, 18.0 ± 2.0% and 2.0 ± 0.4% whereas those on HA-Ti were 17.0 ± 1.2%, 56.0 ± 2.0% and 27.0 ± 1.8%, respectively. The relative OH - concentration on HA-Ti was significantly higher (P b 0.01) than that on Ti. The binding energy values of O1s on HA were 533.4 and 535.1 eV, which were postulated to be PO4 and Ca-OH, respectively. 39 As shown in Figure 2, A, the thickness of a Ti oxide layer was formed on HA-Ti in comparison with Ti. The HA crystals were incorporated within the developing thickness of the Ti oxide layer that was generated during processing. The present high-resolution O1s spectrum on HA-Ti showed a Ti oxide layer rather than HA nanocrystals.
Osteoblast phenotypes on the samples At 3 days of culture, the expressions of Ocn, Osterix, Opn and Bsp2 on HA and HA-Ti were significantly higher (P b 0.01) than those on Ti. The gene expressions in adherent osteoblasts on HA and HA-Ti did not differ significantly (P N 0.01) at 3 days of culture (Figure 3, B). After 7 days of culture (Figure 3, B), the expression of Osterix was still significantly higher (P b 0.01) on HA-Ti than on Ti. The expressions of Opn and Bsp2 on HA and HA-Ti were significantly higher (P b 0.01) than those on Ti, even after 7 days of culture, whereas the expression of Type I collagen on Ti was significantly higher (P b 0.01) than those on HA and HA-Ti. The ALP-positive areas on HA and HA-Ti were greater than that on Ti at 3 days of culture, whereas the areas were similar at 7 days of culture (Figure 3, C). Determination of antimicrobial efficiency The numbers of viable S. mitis and S. gordonii colonies on HA were significantly higher (P b 0.01) than those on HA-Ti and Ti (Figure 4). The numbers of viable S. mitis and S. gordonii on HA-Ti were significantly lower (P b 0.01) than those on HA and Ti. However, the numbers of viable S. mutans and S. sobrinus on the samples were equivalent, with no significant differences (P N 0.01) among the samples.
Discussion HA-Ti showed a developing thickness of Ti oxide film with concomitant growth of hydrophilic functional groups on the surface. Thermally or anodically oxidized Ti surfaces have been reported to show increased surface hydrophilicity because of the formation of OH hydrophilic functional groups 31,33 with the developing thickness of the Ti oxide films. Therefore, the HATi surfaces showed super-hydrophilicity, defined as a contact angle of b 5 degrees, 33 whereas the HA and Ti surfaces exhibited lesser hydrophilicity.
Figure 4. Determination of antimicrobial activity on the samples. Each bacterial cell suspension was prepared at density of 4.4 × 10 3 cells/mL. The entire surface of each sample was prepared with 10 μL of bacteria suspension and covered by polypropylene film. The samples were maintained at room temperature for 6 hrs and then re-immersed in Todd Hewitt broth. In addition, 10 μL of bacteria suspension without cultivation on a sample surface was immersed in Todd Hewitt broth (control). (A) Numbers of viable bacterial cells on the samples after 24 hrs of cultivation. Three samples were evaluated in the antimicrobial tests. The results are expressed as the mean ± SD (n = 3). Data were statistically analyzed by ANOVA followed by a post-hoc Tukey test. A value of P b 0.01 (⁎) was considered significant. (B) Representative pictures of the viable bacterial colonies on Todd Hewitt agar plates after 24 hrs of cultivation.
380
A. Murakami et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 374–382
A. Murakami et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 374–382
Despite its surface super-hydrophilicity, HA-Ti showed a lower cell adhesion property, similar to that observed on HA. The nanoscale HA crystals were incorporated within the developing thickness of the Ti oxide layer on HA-Ti that was generated during processing. A previous study reported that calcium ions were not detectable at the in vitro initial cell adhesion stage on Ti surfaces, whereas the accumulation of sodium or phosphate improved adsorption of noncollagenous serum proteins. 40 Adsorption of noncollagenous serum proteins such as fibronectin or vitronectin on material surfaces plays a critical role in initial cell adhesion to the surfaces. 40 These findings may also imply that local accumulation of calcium ions on the Ti surfaces resisted cell adhesion along with the lesser amount of noncollagenous protein adsorption at the early stage. Because a reduced particle diameter can increase the specific surface area of HA that comes into contact with culture medium or body fluids, the number of cells was distinctively suppressed on HA-Ti rather than on sintered HA. In general, osteoblast differentiation, specializing in mineralization has been evaluated by enhancing the expression of more than 2 types of osteogenic markers in adherent cells to the material surfaces. This was specifically determined by the expression of Ocn, Opn or Bsp 2. Although HA did not show superior initial adhesion properties toward the cells, HA and HATi showed enhanced osteoblast phenotypes in comparison with Ti. Our previous studies indicated that HA specifically conjugates to collagenous matrix proteins rather than noncollagenous serum proteins, such as fibronectin or vitronectin. 35,37 The complex macromolecular assembly of calcium phosphate with extracellular collagenous proteins enables scaffold properties for the adherent osteoblasts. 34,35,37 Thus, it is likely that HA and HA-Ti enhanced the adherent osteoblast phenotypes regardless of the lower cell adhesion properties. Despite the 1-μm thickness of the coating layer, HA-Ti allowed enhanced osteoblast phenotypes, as well as the sintered HA samples, because nanoscale HA can readily conjugate to extracellular collagenous proteins. 41,42 HA-Ti showed antimicrobial activity specifically against early colonizers such as S. mitis and S. gordonii. Only HA-Ti generates an amount of hydrophilic functional groups on the surface because of the thickness of the Ti oxide layer. Therefore, the HA-Ti surface shows a higher negative charge because of these hydrophilic functional groups in comparison with the HA and Ti surfaces. The adhesion mechanisms of the early and late colonizers on the material surfaces have not been fully elucidated. However, because HA-Ti showed antimicrobial activity specifically against S. mitis and S. gordonii, the adhesion mechanisms of these early colonizers may involve an electrostatic force between the material surface and the bacterial cell membranes. On the contrary, the adhesion of the late colonizers S. mutans and S. sobrinus might not involve electrostatic mechanisms. It can be assumed that the antimicrobial activity on HA-Ti was caused by the surface negative charge associated with the OH hydrophilic functional groups, thereby creating a repulsion force between the surface and the bacterial cells. 28 A Ti oxide film formed on Ti surfaces generates hydrophilic functional groups in cell culture conditions, 31 whereas pure HA surfaces exhibit no electrostatic potential. Although the water
381
contact angles on both the HA and bare Ti surfaces did not differ significantly, the Ti surface seemed to generate a repulsion force between the surface and the bacterial membranes, in comparison with the HA surface. The lower electrical charge on the HA surface without hydrophilic functional groups yielded a higher survival rate of adherent early colonizers compared with the HATi and Ti surfaces. Antibiotic-loaded porous HA coatings on Ti have been reported. 17,27 It has been reported that this method leads to burst release of the antibiotics, that is, more than 80–90% of the antibiotic being released from the coating layer within the first 60 minutes. 43,44 Despite the limitation of antimicrobial activity against oral microorganisms, HA-Ti can be expected to ensure longer efficacy rather than antibiotic loading owing to the hydrophilicity. The present findings demonstrated in vitro osteogenic properties, as well as antimicrobial activity, on HA-Ti. HA-Ti has been reported to show in vivo biomechanical integrity because of the micrometer thickness of the functionally graded coating characteristics toward Ti metal substrates. 7,9 Thus, HATi may have potential for use as a surface modification of implantable medical devices, especially dental implants.
References 1. De Groot K, Geesink R, Klein CPAT, Serekian P. Plasma sprayed coatings of hydroxylapatite. J Biomed Mater Res 1987;21:1375-81. 2. Dinda GP, Shin J, Mazumder J. Pulsed laser deposition of hydroxyapatite thin films on Ti-6Al-4V: Effect of heat treatment on structure and properties. Acta Biomater 2009;5:1821-30. 3. Geesink RGT, De Groot K, Klein CPAT. Bonding of bone to apatitecoated implants. J Bone Joint Surg - Series B 1988;70:17-22. 4. Gotfredsen K, Wennerberg A, Johansson C, Skovgaard LT, HjortingHansen E. Anchorage of TiO2-blasted, HA-coated, and machined implants: An experimental study with rabbits. J Biomed Mater Res 1995;29:1223-31. 5. Le Guehennec L, Soueidan A, Layrolle P, Amouriq Y. Surface treatments of titanium dental implants for rapid osseointegration. Dent Mater 2007;23:844-54. 6. Tsui YC, Doyle C, Clyne TW. Plasma sprayed hydroxyapatite coatings on titanium substrates. Part 1: Mechanical properties and residual stress levels. Biomaterials 1998;19:2015-29. 7. Shibata Y, Takashima H, Yamamoto H, Miyazaki T. Functionally gradient bonelike hydroxyapatite coating on a titanium metal substrate created by a discharging method in HBSS without organic molecules. Int J Oral Maxillofac Implants 2004;19:177-83. 8. Wheeler SL. Eight-year clinical retrospective study of titanium plasmasprayed and hydroxyapatite-coated cylinder implants. Int J Oral Maxillofac Implants 1996;11:340-50. 9. Yamamoto H, Shibata Y, Tachikawa T, Miyazaki T. In vivo performance of two different hydroxyapatite coatings on titanium prepared by discharging in electrolytes. J Biomed Mater Res - Part B Applied Biomaterials 2006;78:211-4. 10. Zheng X, Huang M, Ding C. Bond strength of plasma-sprayed hydroxyapatite/Ti composite coatings. Biomaterials 2000;21:841-9. 11. Albrektsson T, Sennerby L, Wennerberg A. State of the art of oral implants. Periodontology 2000 2008;47:15-26. 12. Chang JK, Chen CH, Huang KY, Wang GJ. Eight-Year Results of Hydroxyapatite-Coated Hip Arthroplasty. J Arthroplasty 2006;21:541-6. 13. Geesink RGT, Hoefnagels NHM. Six-year results of hydroxyapatitecoated total hip replacement. J Bone Joint Surg - Series B 1995;77: 534-47.
382
A. Murakami et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 374–382
14. Callan DP, Cobb CM, Williams KBDNA. probe identification of bacteria colonizing internal surfaces of the implant-abutment interface: A preliminary study. J Periodontol 2005;76:115-20. 15. Omori S, Shibata Y, Arimoto T, Igarashi T, Baba K, Miyazaki T. Microorganism and cell viability on antimicrobially modified titanium. J Dent Res 2009;88:957-62. 16. Deng JY, Arimoto T, Shibata Y, Omori S, Miyazaki T, Igarashi T. Role of chloride formed on anodized titanium surfaces against an oral microorganism. J Biomater Appl 2010;25:179-89. 17. Zhao L, Chu PK, Zhang Y, Wu Z. Antibacterial coatings on titanium implants. J Biomed Mater Res-Part B Appl Biomater 2009;91:470-80. 18. Gao Y, Zou S, Liu X, Bao C, Hu J. The effect of surface immobilized bisphosphonates on the fixation of hydroxyapatite-coated titanium implants in ovariectomized rats. Biomaterials 2009;30:1790-6. 19. O'Hare P, Meenan BJ, Burke GA, Byrne G, Dowling D, Hunt JA. Biological responses to hydroxyapatite surfaces deposited via a coincident microblasting technique. Biomaterials 2010;31:515-22. 20. Saber-Samandari S, Gross KA. The use of thermal printing to control the properties of calcium phosphate deposits. Biomaterials 2010;31:6386-93. 21. Slots J, Listgarten MA. Bacteroides gingivalis, bacteroides intermedius and actinobacillus actinomycetemcomitans in human periodontal diseases. J Clin Periodontol 1988;15:85-93. 22. Moore WE. Microbiology of periodontal disease. J Periodont Res 1987;22:335-41. 23. Medaglini D, Pozzi G, King TP, Fischetti VA. Mucosal and systemic immune responses to a recombinant protein expressed on the surface of the oral commensal bacterium Streptococcus gordonii after oral colonization. Proc Nat Acad Sci U S A 1995;92:6868-72. 24. Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL. Microbial complexes in subgingival plaque. J Clin Periodontol 1998;25:134-44. 25. Ge X, Leng Y, Bao C, Xu SL, Wang R, Ren F. Antibacterial coatings of fluoridated hydroxyapatite for percutaneous implants. J Biomed Mater Res-Part A 2010;95 A:588-99. 26. Mo A, Liao J, Xu W, Xian S, Li Y, Bai S. Preparation and antibacterial effect of silver-hydroxyapatite/titania nanocomposite thin film on titanium. Appl Surf Sci 2008;255:435-8. 27. Stigter M, Bezemer J, De Groot K, Layrolle P. Incorporation of different antibiotics into carbonated hydroxyapatite coatings on titanium implants, release and antibiotic efficacy. J Control Release 2004;99:127-37. 28. Chua PH, Neoh KG, Kang ET, Wang W. Surface functionalization of titanium with hyaluronic acid/chitosan polyelectrolyte multilayers and RGD for promoting osteoblast functions and inhibiting bacterial adhesion. Biomaterials 2008;29:1412-21. 29. Zhitomirsky I, Gal-Or L. Electrophoretic deposition of hydroxyapatite. J Mater Sci: Mater Med 1997;8:213-9.
30. Ma J, Wang C, Peng KW. Electrophoretic deposition of porous hydroxyapatite scaffold. Biomaterials 2003;24:3505-10. 31. Zhao G, Schwartz Z, Wieland M, Rupp F, Geis-Gerstorfer J, Cochran DL, et al. High surface energy enhances cell response to titanium substrate microstructure. J Biomedl Mater Res - Part A 2005;74:49-58. 32. Park YJ, Song HJ, Kim I, Yang HS. Surface characteristics and bioactivity of oxide film on titanium metal formed by thermal oxidation. J Mater Sci Mater Med 2007;18:565-75. 33. Shibata Y, Suzuki D, Omori S, Tanaka R, Murakami A, Kataoka Y, et al. The characteristics of in vitro biological activity of titanium surfaces anodically oxidized in chloride solutions. Biomaterials 2010;31: 8546-55. 34. Shibata Y, He LH, Toda Y, Kataoka Y, Fujisawa N, Miyazaki T, et al. Micromechanical evaluation of mineralized multilayers. J Biomech 2008;41:3414-8. 35. Toda Y, Shibata Y, Kataoka Y, Kawawa T, Miyazaki T. Interfacial assembly of bioinspired nanostructures mediated by supersensitive crystals. J Biomed Mater Res-Part A 2008;84:869-74. 36. Takashima H, Shibata Y, Kim TY, Miyazaki T. Hydroxyapatite coating on a titanium metal substrate by a discharging method in modified artificial body fluid. Int J Oral Maxillofac Implants 2004;19: 66-72. 37. Shibata Y, Yamamoto H, Miyazaki T. Colloidal beta-tricalcium phosphate prepared by discharge in a modified body fluid facilitates synthesis of collagen composites. J Dent Res 2005;84:827-31. 38. Suda T, Jimi E, Nakamura I, Takahashi N. Role of 1 alpha,25dihydroxyvitamin D3 in osteoclast differentiation and function. Methods Enzymol 1997;283:223-35. 39. Santos JD, Jha LJ, Monteiro FJ. In vitro calcium phosphate formation on SiO2-Na2O-CaO-P2O5 glass reinforced hydroxyapatite composite: A study by XPS analysis. J Mater Sci Mater Med 1996;7:181-5. 40. Yamamoto H, Shibata Y, Miyazaki T. Anode glow discharge plasma treatment of titanium plates facilitates adsorption of extracellular matrix proteins to the plates. J Dent Res 2005;84:668-71. 41. Chang MC, Tanaka J. FT-IR study for hydroxyapatite/collagen nanocomposite cross-linked by glutaraldehyde. Biomaterials 2002;23: 4811-8. 42. Chang MC, Tanaka J. XPS study for the microstructure development of hydroxyapatite-collagen nanocomposites cross-linked using glutaraldehyde. Biomaterials 2002;23:3879-85. 43. Radin S, Campbell JT, Ducheyne P, Cuckler JM. Calcium phosphate ceramic coatings as carriers of vancomycin. Biomaterials 1997;18:777-82. 44. Yamamura K, Iwata H, Yotsuyanagi T. Synthesis of antibiotic-loaded hydroxyapatite beads and in vitro drug release testing. J Biomed Mater Res 1992;26:1053-64.