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Biomacromolecules 2010, 11, 1254–1260
Surface Functionalization of Titanium with Chitosan/Gelatin via Electrophoretic Deposition: Characterization and Cell Behavior Tao Jiang,†,‡ Zhen Zhang,†,‡ Yi Zhou,‡ Yi Liu,‡ Zhejun Wang,‡ Hua Tong,§ Xinyu Shen,§ and Yining Wang*,‡ Key Laboratory for Oral Biomedical Engineering, Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079, People’s Republic of China, and Institute of Analytical and Biomedical Sciences, School of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, People’s Republic of China Received January 15, 2010; Revised Manuscript Received March 25, 2010
The electrophoretic deposition (EPD) is a versatile and cost-effective technique for fabricating advanced coatings. In this study, chitosan/gelatin (CS/G) coatings were prepared on titanium substrates via EPD. The prepared coatings were characterized using fluorescence microscopy, scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and shear bond strength testing. It was found that CS/G coatings had a similar macroporous structure. The gelatin content in the CS/G coatings gradually increased with the increase of the gelatin in the blend solutions. The shear bond strength of the CS/G coatings also increased with the increasing gelatin content. In vitro biological tests demonstrated that human MG63 osteoblast-like cells achieved better affinity on the coatings with higher gelatin content. Therefore, it was concluded that EPD was an effective and efficient technique to prepare CS/G coatings on the titanium surface and that CS/G coatings with higher gelatin content were promising candidates for further loading of functional agents.
Bone-anchored endosseous titanium implants have been increasingly used in craniofacial, dental, and orthopedic surgery since the pioneering work of Brånemark.1 The clinical success of titanium implants is attributed to their ability of osseointegration, which is defined as a direct and stable anchorage of an implant by the formation of bony tissue without growth of fibrous tissue at the bone-implant interface.2 Despite its great success in the clinical application, there are still challenges for designing successful implants because of poor bone quality and prevention of acute inflammation around the implant.3 Because the surface properties of titanium implants play important roles in osseointegration, a variety of strategies of modifying implant surfaces have been developed to induce controlled, guided, and rapid osseous healing,4-7 including alteration of surface physicochemical, morphological, and biochemical properties of titanium implant.8-10 Chitosan is a cationic natural polymer that carries positive charges with a molecular structure of (1,4)-linked 2-amino-2deoxy-β-D-glucan. It has been widely applied in the biomedical field, such as wound healing, drug delivery, and tissue engineering due to its flexibility, biocompatibility, biodegradability, antibacterial property, and low cost.11,12 Chitosan has gained considerable attentions for its use as a coating material on titanium implants in recent years. It was reported that chitosan could be coated on titanium via solution casting,13 silane reactions,14 or layer-by-layer technique.15 Wu et al.16,17 found that chitosan could be deposited onto the surface of a negative Au electrode via electrophoretic deposition (EPD). This led to the speculation that chitosan coatings could be prepared on
titanium substrate via EPD. Compared with those techniques mentioned above, EPD has advantages of short processing time, simple producing apparatus, and no requirement for cross-link agents. Complex fabricated objects can also be easily coated on the inside cavities as well as outside surfaces. In particular, as a wet process, EPD offers easy control of the thickness and morphology of a deposited coating through simple adjustment of the deposition time and applied potential.18 Hence, we prepared chitosan coating on titanium substrate via EPD. However, cell affinity of the pure chitosan coating was not as good as what we expected. This finding was supported by several other studies that showed unsatisfactory osteoblastic cell compatibility of chitosan.19,20 It has been reported that chitosan could mediate particles deposition, which provides a simple approach to assemble particles at addressable locations.17,21,22 The possibility of codeposition of chitosan and other functional materials opens new opportunities in the fabrication of advanced coatings on titanium implant.23 Gelatin is a natural macromolecular material that is derived from collagen and has been widely used as food ingredients, pharmaceutical capsules, and biomedical tissue engineering.24 Gelatin is a biodegradable polymer with many attractive properties, such as excellent biocompatibility, nonantigenicity, plasticity, and adhesiveness.25,26 Moreover, gelatin can form a polyelectrolyte complex with chitosan at suitable pH value.23,27 Therefore, we hypothesized that chitosan and gelatin could be codeposited onto the titanium via EPD and the addition of gelatin could improve the mechanical and biological properties of the coatings.
* To whom correspondence should be addressed. Tel.: +86 27 87686318. Fax: +86 27 87873260. E-mail:
[email protected]. † Contributed equally to this work. ‡ School and Hospital of Stomatology, Wuhan University. § School of Chemistry and Molecular Sciences, Wuhan University.
Materials. Chitosan (Mw 1000000, humidity 7.86%, and ash content 0.80%) with a deacetylation degree greater than 95% (Golden-Shell
Introduction
Materials and Methods
10.1021/bm100050d 2010 American Chemical Society Published on Web 04/02/2010
Surface Functionalization of Titanium Biochemical Co., Ltd.) and gelatin (type A) derived from acid-cured tissue (Sigma, G1890) were used as received. Commercial pure titanium (grade 2) was supplied by Baoji Titanium industry Co., Ltd. Loctite 454 instant gel adhesive was purchased from Henkel Loctite Corp (Loctite, Rock Hill, CT). Human MG63 osteoblast-like cells (ATCC catalog CRL-1427) were obtained from American Type Culture Collection. All the other chemicals reagents were local products of analytical grade. Titanium Substrates Preparation. Titanium pieces were cut into rectangular plates (20 × 10 × 1 mm) for shear bond strength testing or turned into disks (15 mm in diameter, 1 mm thick) for cell culture in 24-well cell culture plate. All the plates and disks were treated as described in our previous article.28 In brief, the specimens were coarse grit blasted with 0.25-0.50 mm corundum grit at 5 bar for 1 min, and afterward, acid-etched in hydrochloric acid/sulfuric acid (1:1) at 65 °C for 30 min. After that, they were ultrasonically cleaned with acetone, ethanol, and Milli-Q water for 20 min each procedure and then rinsed with Milli-Q water. EPD Process. Chitosan solution was obtained by dissolving 1.2 mg chitosan in 150 mL of 0.04 M HCl solution. The blend solutions of chitosan and gelatin were prepared by dissolving different amounts of gelatin powder into chitosan solutions at 60 °C for 2 h with magnetic stirring. Pure chitosan solution was also treated in the same way. All solutions were adjusted to pH 4.0 using 0.1 M NaOH and then brought to a total volume of 200 mL with Milli-Q water and stirred with magnetic bar for 24 h at room temperature. Blends containing 30, 50, and 70 wt % gelatin were produced and coded here as CS/G30, CS/ G50, and CS/G70, respectively. During EPD process, Ti disk or plate was used as cathode and parallel platinum plate as counterelectrode. The distance between the cathode and counterelectrode was 50 mm. Deposition was performed by connecting both the cathode and the anode to a direct current power supply (Model 6614C, Agilent Technologies) with a constant voltage of 3.5 V for 3 min. After deposition, the electrodes were disconnected from the power supply, removed from the solution, rinsed with Milli-Q water, and finally dried at 37 °C in water interlining thermostatic culturing cabinet (Shanghai Yiheng Technical Co., Ltd.) overnight. Characterization. The optical photographs of the coatings were taken immediately after EPD. After that, the coatings were fluorescently labeled with acridine orange (Sigma, A6014) and visualized by inverted fluorescence microscopy (Nikon TE-2000) with a CCD camera (Spot Diagnostic Instruments Inc.). Surface morphology of the coatings was observed by scanning electron microscope (SEM; Fei Quanta-200, Netherlands). The surface chemistry of the coatings was investigated with attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR; Thermo Nicollet 5700, U.S.A.). X-ray diffraction pattern (XRD) of the coatings was measured using an X-ray Diffractometer (Bruker Axs D8 Advance, Germany) under a voltage of 30 kV and 30 mA using Cu KR radiation. The diffraction pattern was determined over a range of diffraction angle 2θ ) 5° to 2θ ) 40° at a rate of 1° (2θ) per min and a step size of 0.1° (2θ). Shear Bond Strength. To investigate the coating-titanium interface, the shear bond strength was tested according to the standard method (ASTM: D 1002-05) with an electrical mechanical Instron Model 4465 load frame (Instron Corporation, Norwood, MA) with a 5000 N load cell. The coating surface was adhered to another titanium plates using instant gel adhesive, dead weighted to ensure contact, and cured for 24 h at ambient temperature. The pull test was run at a constant crosshead displacement of 0.50 mm per min until failure was reached as it was evidenced by a drop in load. Five samples per group were used for statistical analysis. Cell Culture. MG63 osteoblast-like cells were cultured in Dulbecco’s modified Eagle medium (DMEM) in the presence of 10% fetal bovine serum (FBS) at 37 °C in a humidified 5% CO2 atmosphere. When cells reached 80-90% confluence, they were trypsinized and suspended in the culture medium. Cell count and viability were carried out using the Beckman-Coulter automatic cell counter (VI-cell analyzer,
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Beckman Coulter, Inc.). All disks were steam sterilized and then placed in 24-well tissue culture plates under aseptic conditions. After that, they were seeded at a cell density of 1000 cells/cm2. The culture medium was renewed every three days. Observation of Cell and Coatings In Situ. After 7 days of culture, some disks were gently rinsed with PBS for three times. Then they were fixed in 95% alcohol for 10 min at 4 °C. After being thoroughly washed with PBS, the samples were stained with 0.025% acridine orange solution for 5 min and then rinsed with 1% calcium chloride solution for 1 min. Fluorescence images were obtained using a Nikon TE-2000 inverted microscope. Cell Morphology and Skeleton. For cell morphological observation, some disks were examined by SEM. After 2 days of culture, the disks were gently washed with PBS for 3 times. Then they were fixed with 2.5% glutaraldehyde in sodium dimethylarsenate solution for 1 h at 4 °C. After being thoroughly washed with PBS, the samples were dehydrated through a series of graded ethanol. After that, they were gold sputter coated (around 10 nm) in vacuum and examined by SEM. For cell skeleton observation, some samples were fixed with 3.7% paraformaldehyde for 10 min, washed with PBS, and permeabilized with 0.1% Triton X-100 solution for 5 min. The nonspecific binding sites were blocked by incubating the coatings in PBS containing 1% bovine serum albumin for 30 min. Filamentous actins (F-actins) were stained with rhodamine phalloidin (R-415 kit, Molecular Probes, Invitrogen, U.S.A.) for 20 min at room temperature. The nuclei were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; 1:1000 dilutions in PBS; Invitrogen, Basel, Switzerland) for 15 min at room temperature. The samples were rinsed again with PBS and then stored in PBS. Immunofluorescence images were obtained using a Nikon TE-2000 inverted microscope. The fluorescence images with two colors were taken by double exposure with red light exposure first and blue next. Cell Proliferation. Cell proliferation was quantitatively analyzed by using cell counting kit-8 (CCK8; Dojindo Laboratories, Japan). After 1, 3, and 7 days of culture, some disks were gently washed with PBS and then 0.5 mL of DMEM containing 10% CCK-8 was added per well. The disks were incubated at 37 °C for 3 h. The absorbance of supernatant was then measured at 450 nm using an ELX808 Ultra Microplate Reader (Bio-Tek Instruments, Inc., U.S.A.). The optical density values were determined at least in triplicate. The values reflected the viable cell population in each well. Statistical Analysis. Quantitative data were expressed as means ( standard deviation (SD). A one-way analysis of variance (ANOVA) was used to determine statistical significance followed by post hoc analysis using the Tukey test. The level of significance was set at a P value of 0.05.
Results Topography Description. The optical photographs showed that the coatings presented gelatinous structure on titanium substrates (Figure 1a-d). Fluorescence images presented that all coatings had similar macroporous structure (Figure 1f-i). The pore size ranged from 50 to 200 µm. Low magnification SEM micrographs showed that the pore walls of pure chitosan coating were relatively intact (Figure 2a). Some cracks and fissures appeared on that of CS/G30 coating (Figure 2b) and fragmented wall structure presented on those of CS/G50 and CS/G 70 coatings (Figure 2c,d). High magnification SEM micrographs displayed that the coatings’ surface gradually became smooth from pure chitosan to CS/G 70 coating (Figure 2). ATR-FTIR Analysis. ATR-FTIR spectrum of the pure chitosan coating displayed strong peaks at 1151, 1083, and 1033 cm-1 (Figure 3a), which were characteristic peaks of a polysaccharide structure due to C-N stretching, C-O stretching, and O-H bending.29 The spectrum of gelatin, which was coated on titanium substrate by evaporation, showed the strong
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Figure 1. Photographs and fluorescence images of pure chitosan and CS/G coatings: (a,f) pure chitosan coating; (b,g) CS/G30 coating; (c,h) CS/G50 coating; (d,i) CS/G70 coating; and (e,j) titanium.
Figure 2. SEM micrographs of pure chitosan and CS/G coatings: (a) pure chitosan coating; (b) CS/G30 coating; (c) CS/G50 coating; (d) CS/ G70 coating; and (e) titanium.
absorption peaks at 1638 and 1548 cm-1 due to amide I, -CONH- stretching, and amide II, -NH2 stretching, respectively.30 These two peaks gradually increased from CS/G30 to CS/G70 coating and shifted to higher wavenumbers at 1650-1642 and 1552-1500 cm-1, respectively (Figure 3b-d). XRD Analysis. The XRD patterns of chitosan powder displayed diffraction peaks at 2θ ) 11.8° and 2θ ) 20.4° (Figure 4f). The pattern of pure chitosan coating exhibited that the peak at 2θ ) 11.8° was indistinct, while the peak at 2θ ) 20.4° became broad (Figure 4a). The patterns of CS/G30, CS/ G50, and CS/G70 coatings were similar to that of pure chitosan coating (Figure 4b-d). The reflection peaks at 2θ ) 35.1, 38.4, and 40.2° corresponded to the titanium substrate. Shear Bond Strength. The testing relies on the bonding agent to remove the coating from titanium substrate by applying shear strength. It was found that all coatings were detached from titanium substrate after testing. The failures occurred at the coating-titanium interface rather than coating-gel adhesive interface. The mean bond strength of pure chitosan, CS/G30, CS/G50 and CS/G70 coatings were 4.12, 6.56, 7.28, and 8.01 MPa, respectively. Statistical analysis indicated that the bond strength of the CS/G70 coating was significantly greater than that of the pure chitosan coating (p < 0.05). There was no
Figure 3. ATR-FTIR spectra of pure chitosan and CS/G coatings: (a) pure chitosan coating; (b) CS/G30 coating; (c) CS/G50 coating; (d) CS/G70 coating; and (e) gelatin film.
statistical difference among those of CS/G30, CS/G50, and CS/ G70 coatings (Figure 5).
Surface Functionalization of Titanium
Figure 4. XRD patterns of pure chitosan and CS/G coatings: (a) pure chitosan coating; (b) CS/G30 coating; (c) CS/G50 coating; (d) CS/ G70 coating; (e) titanium; and (f) chitosan powder.
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pure chitosan and CS/G30 coatings (Figure 6a,b), while large numbers of cells homogeneously distributed on CS/G50 and CS/G70 coatings and titanium substrate (Figure 6c-e). The nuclei of cells cultured on pure chitosan and CS/G30 coatings exhibited a bright orange-red fluorescence, which meant that they were apoptotic cells. Cell Morphology and Skeleton. SEM micrographs showed that cells displayed a roughly spherical morphology with a diameter of 10 µm on pure chitosan and CS/G30 coatings (Figure 7a,b), while cells presented polygonal morphology and spread extensively on CS/G50 coating, CS/G70 coating, and titanium substrate. (Figure 7c-e). Immunofluorescence images revealed the information of actin cytoskeleton, which provides structural framework and participates in cell migration.31 In contrast to the hardly visible filopodia of cells on pure chitosan and CS/G30 coatings (Figure 8a,b), cells had bright and well-pronounced elongated filopodia projected from the cell edges on CS/G50 and CS/G70 coatings (Figure 8c,d). There were some membrane channels between adjacent cells on CS/G50 and CS/G70 coatings (Figure 8c,d). These actin-rich structures are termed as tunneling nanotubes,32 which can mediate the intercellular transfer of organelles, plasma membrane components, and cytoplasmic molecules.33 Cell Proliferation. After 1 day of culture, CCK-8 counts of cells displayed no significant difference among various coatings and titanium substrate (Figure 9). After 3 days of culture, CCK-8 counts of CS/G50 and CS/G70 coatings were significantly higher than those of pure chitosan and CS/G30 coatings (p < 0.05). After 7 days of culture, CCK-8 counts increased from pure chitosan to CS/G70 coating. It was noted that the CS/G70 coatings had the similar CCK-8 counts with titanium substrate on days 3 and 7.
Discussion
Figure 5. Bar graph of the shear bond strengths of pure chitosan and CS/G coatings on titanium substrate. Error bars represent means ( SD for n ) 5 (*p < 0.05).
Cell Growth on Coatings. In situ observation of cells and coatings revealed that only a few cells located in the pores of
In this study, pure chitosan and CS/G coatings were successfully prepared on titanium substrates via EPD. The results supported our original hypothesis that chitosan and gelatin could be codeposited onto the titanium via EPD. It should be mentioned, however, that pure gelatin could not be deposited without the mediation of chitosan (data not shown). It was found that the coatings presented macroporous structure with the pore size ranging from 50 to 200 µm. This should be attributed to the gas evolution at the electrode, which caused
Figure 6. Acridine orange fluorescence staining of cells cultured on pure chitosan and CS/G coatings: (a) pure chitosan coating; (b) CS/G30 coating; (c) CS/G50 coating; (d) CS/G70 coating; and (e) titanium. A few cells located in the pores of pure chitosan and CS/G30 coatings (arrow), while large numbers of cells distributed uniformly on CS/G50, CS/G70 coatings, and titanium substrate.
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Figure 7. SEM micrographs of cell morphology on pure chitosan and CS/G coatings: (a) pure chitosan coating; (b) CS/G30 coating; (c) CS/G50 coating; (d) CS/G70 coating; and (e) titanium.
Figure 8. Fluorescence images of cells skeletons: (a) pure chitosan coating; (b) CS/G30 coating; (c) CS/G50 coating; (d) CS/G70 coating; and (e) titanium. MG63 cells attached to CS/G50 and CS/G70 coatings displayed more bundles (arrow) of actin microfilaments than pure chitosan and CS/G30 coatings.
the air bubbles to be trapped in the deposited coatings during EPD procedure.18 It has been reported that large pore size of biomaterial scaffolds resulted in greater bone ingrowths in vivo.34 Therefore, it is reasonable to expect that these macroporous coatings may have satisfactory performance in vivo. The ATR-FTIR results proved that gelatin was incorporated into coatings with the deposition of chitosan. The increase in absorption peaks of -CONH- and -NH2 indicated that the gelatin content in the coatings gradually increased with the increase of the gelatin in the blend solutions.35 The shifting of these two vibration peaks indicated that there was a kind of chemical interaction between gelatin and chitosan.36 A characteristic feature of the EPD process in this study was that colloidal chitosan and gelatin particles suspended in a liquid medium migrated and were deposited onto the titanium substrate
under the influence of an electric field. Chitosan is a linear polysaccharide, which has primary amino groups with pKa values around 6.3.37 If the pH below the pKa, chitosan will be soluble and most of its amino groups will be protonated (Figure 10a).16 When gelatin was dissolved in chitosan solution, the colloidal polyelectrolyte complexes were formed by the electrostatic interaction between -NH3+ groups carried on chitosan and -COO- groups on gelatin (Figure 10b).27 After voltage was applied between two electrodes for a certain time, chitosan experienced a higher pH than pKa near the cathode, where chitosan’s amino groups were deprotonated and became insoluble. Then, chitosan and gelatin were codeposited onto the surface of titanium substrates (Figure 10c). The XRD patterns indicated that chitosan in all coatings was predominantly in amorphous form. The decreased structural
Surface Functionalization of Titanium
Figure 9. CCK-8 counts of cells cultured on pure chitosan and CS/G coatings. The initial seeding density was 1000 cells/cm2. Error bars represent means ( SD for n ) 4 (*p < 0.05, **p < 0.01).
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et al.41 reported that the inhibition of cell proliferation on chitosan coatings was due to reduction of cell adhesion. Another explanation could be that glucosamine, which randomly distributed in linear polysaccharide of chitosan, reduced the metabolism of MG63 cells.42 In contrast to our study, several studies found that chitosan supported the initial attachment, spreading, and growth of osteoblasts.43,44 The discrepancy is probably due to the variation in properties of chitosan, such as degree of deacetylation, molecular weight, initial source material, production lots, and so on.11,20 In vitro biological tests showed that CS/G coatings with higher gelatin content achieved better cell response as compared to pure chitosan coating, which confirmed our hypothesis that the incorporation of gelatin could enhance the cell affinity of coatings. This could be explained by the fact that gelatin contains Arg-Gly-Asp (RGD) like sequences, which promote cell adhesion, migration, and proliferation.41,45 Moreover, the electrostatic interaction of gelatin and chitosan might decrease the charge density of the coatings,19 then the cell proliferation was enhanced by the resultant weakening electrostatic effects between cell membranes and the coatings. In this study, chitosan and gelatin were shown to complement each other in the CS/G coating, namely, chitosan mediated the deposition of gelatin, while gelatin enhanced the cell biocompatibility and shear bond strength of the coatings. Because CS/G coatings with higher gelatin content (CS/G50 and CS/G70) showed good cell biocompatibility, they could be promising candidates for further loading of functional agents.
Conclusions In this paper, pure chitosan and CS/G coatings were successfully prepared on titanium substrates via EPD. It was found that all coatings had similar macroporous structure. The gelatin content incorporated into coatings gradually increased with the increase of the gelatin in the blend solutions. The addition of gelatin not only increased the shear bond strength of the coatings but also improved the biological response of osteoblastic cells. The possibility of codeposition of CS/G coatings with other functional agents, such as anticancer, antibiotics, proteins, gene segment, and amino acid, provides new opportunities in the fabrication of functional coatings on titanium substrate.
Figure 10. Schematic representation of the mechanism of CS/G coating deposited onto titanium substrate. (a) Chitosan was protonated in hydrochloric acid solution; (b) gelatin formed polyelectrolyte complex with chitosan; and (c) CS/G coating were deposited onto the titanium substrate.
order of chitosan might result from the solution preparation38,39 and EPD procedure.21,40 It has been reported that production of cast chitosan film resulted in a decrease of the crystallinity.38,39 In addition, Zhitomirsky et al.21,40 found that the chitosan in composite coatings, which was prepared via EPD, was also in amorphous form. The results of acridine orange staining, cell morphology, and proliferation demonstrated that pure chitosan coating restricted cell spreading and proliferation. Currently, it is still unclear what biochemical events make chitosan be cytostatic toward. One possible reason is the strong electrostatic interactions between the negative charges of the surface of cell membranes and the cationic sites on the chains of chitosan.19 Nevertheless, Huang
Acknowledgment. We gratefully acknowledge the financial support from National Natural Science Foundation of China (No. 30872910), National Key Technology R&D Program of China during the 11th Five-Year Plan (No. 2007BAI18B05), and SelfResearch Program for Doctoral Candidates of Wuhan Unversity in 2008.
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BM100050D
