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Mar 21, 2016 - report in situ formation of chitosan microtube (CMT) structures from chitosan films on the implant surface with titania nanotubes (TNTs...
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In Situ Transformation of Chitosan Films into Microtubular Structures on the Surface of Nanoengineered Titanium Implants Karan Gulati,† Lucas Johnson,†,‡ Ramesh Karunagaran,†,‡ David Findlay,§ and Dusan Losic*,† †

School of Chemical Engineering and §Discipline of Orthopaedics and Trauma, University of Adelaide, Adelaide, SA, Australia S Supporting Information *

ABSTRACT: There is considerable interest in combining bioactive polymers such as chitosan with titanium bone implants to promote bone healing and address therapeutic needs. However, the fate of these biodegradable polymers especially on titanium implants is not fully explored. Here we report in situ formation of chitosan microtube (CMT) structures from chitosan films on the implant surface with titania nanotubes (TNTs) layer, based on phosphate bufferinduced transformation and precipitation process. We have comprehensively analyzed this phenomenon and the factors that influence CMT formation, including substrate topography, immersion solution and its pH, effect of coating thickness, and time of immersion. Significance of reported in situ formation of chitosan microtubes on the TNTs surface is possibly to tailor properties of implants with favorable micro and nano morphology using a self-ordering process after the implant’s insertion.

1. INTRODUCTION There are a large number of disease conditions that would greatly benefit from the ability to deliver drugs locally, in a controlled fashion. These include skeletal conditions, such as infection, bone tumors, local osteoporosis, and hard-to-heal fractures.1 Systemic administration of drugs is often used to address these problems, but this approach has limitations, including low drug solubility, poor bioavailability, uncontrolled pharmacokinetics, and toxicity to non-target tissues.2 In contrast, localized delivery of drugs from the implants such as titanium with electrochemically engineered oxide layer composed of vertically aligned array of titania nanotubes (TNTs) potentially provides a feasible solution to the abovementioned challenges.3 To improve drug releasing performance of these implants, many structural and surface modification strategies have been suggested, including controlling nanotube dimensions and shapes, surface functionalization and biopolymer coating.4−6 The most appealing were biopolymer coatings such as chitosan, PLGA [poly(lactic-co-glycolic acid)] and gelatin, showing favorable release kinetics with extended drug release, controlled by the rate of polymer consumption (in the presence of releasing medium) and thickness of polymer layer.2 Natural biopolymer, chitosan in particular has attracted interest for bone tissue engineering and orthopedic implant applications, due to its ability to be processed into fibers, beads and coatings that can be loaded with active therapeutics, and its inherent antibacterial and osseo-integrating properties.7−9 These unique properties are a consequence of its resemblance to the molecular structure of glycosaminoglycan, a primary component of the extracellular matrix or ECM.10,11 Furthermore, chitosan is readily obtained from chitin, a structural © 2016 American Chemical Society

component of crustaceans, insects and the cell-walls of bacteria/fungi, which makes it the second most abundant organic compound known, and hence economical to produce.12 In addition, its suitability for implant modifications arises from its non-toxicity, biodegradability, biocompatibility, and proven antifungal and antibacterial properties.8 On the other hand, successful fabrication of TNTs on complex substrate geometries such as needles, wires, meshes, plates, screws, etc. opens the exciting possibility to integrate this technology into existing orthopedic implants.13 TNTs on Ti wires have been explored previously by our group to demonstrate their advantages such as minimally invasive drug-releasing implants for potential localized bone and cancer therapies.14−16 Furthermore, these nanoengineered implants, in the form of tiny wires or pins, have been demonstrated to release drug three-dimensionally inside the bone ex vivo and, hence, have been proposed as “fit and forget” therapeutic bone implants.15 In our previous study we have shown that the drug-loaded TNT/Ti implants coated with chitosan film not only reduced the initial burst release and delayed the overall release for >4−6 weeks, but also simultaneously enhanced the bone cell adhesion and antibacterial property of the TNT implants.17,18 These properties are highly dependent on the dissolution rates of chitosan, which is expected to be influenced by many parameters including topography and catalytic properties of TNTs, type of drug, thickness of chitosan layer, temperature, Received: August 1, 2015 Revised: February 9, 2016 Published: March 21, 2016 1261

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Scheme 1. Schematic Showing In Situ Transformation of Chitosan Film Deposited on Titania Nanotube Surface (TNT Wire Implant) and the Formation of Chitosan Microtube (CMT) Structures Based on Phosphate-Buffer-Induced Neutralization Process

2. EXPERIMENTAL SECTION

pH, presence of other molecules in environment able to react with chitosan, and so on. However, in-depth knowledge of the consumption/precipitation behavior of chitosan coatings on TNT implants is not well understood and demands further investigation. Addressing this research gap is critical especially for the long-term osseo-integration of implants. For instance, having favorable microscale architecture on/with nanotubular topography can provide enhanced bone cell functions and ultimately lead to improved bone healing rates.19,20 This in turn can promote bone cell adhesion and influence cell differentiation/proliferation abilities.20 Therefore, there is a significant advantage to chitosan coatings on bone implants having a microfibrous architecture, as this will potentially help ensure long-term osseo-integration. Several techniques have been explored to achieve this topography including phase separation, electro-spinning and template-assisted, which have shown upregulated bone cell functions both in in-vitro and in-vivo settings.21−23 The formation of chitosan tubes/fibers on Ti implants, using electrospinning has shown the most promise, as it permits easy control over the fiber dimensions and gives even surface coverage, while retaining the antibacterial property of the bulk chitosan.21,24 However, electrospinning and other fabrication procedures require expensive equipment or chemical/temperature treatments, and are generally applicable for implant modification prior to surgical placement inside the traumatized tissue such as fractured bone. To address these limitations, here we describe the formation of unique hollow chitosan microtubes (CMTs) on the TNT/Ti surface created by precipitation/neutralization of chitosan film when exposed to phosphate buffered saline (PBS; pH 7.4). The process is schematically outlined in Scheme 1. Chitosan film on the surface of TNT/Ti wire is formed by simple dip coating method followed by immersion in PBS solution, which is isotonic to various human fluids. We propose that chitosan film on TNT implants upon surgical placement inside the target tissue, in the presence of physiological fluids, will be transformed into microstructures, thereby creating a dualtopography with combined micro- and nano-range roughness. This concept for the first time shows the possibility that favorable mixed micro- and nanotopography on implant surface can be created in situ after insertion of the implant and significantly improve its bioactivity, bone healing, and integration properties. A comprehensive study of this process is presented to explore the factors influencing the chitosan microtube formation, including the substrate topography, type of immersion solution, pH, number of chitosan coatings, and immersion time.

Materials. High purity titanium wires (diameter 0.50 mm) were supplied from Nilaco (Japan). Ethylene glycol, ammonium fluoride (NH4F), and chitosan (low molecular weight: 50k−190k Daltons, deacetylation: ≥75%) were obtained from Sigma-Aldrich (Sydney, Australia). High purity water Option Q-Purelabs (Australia; 18.2 MΩ) was used for preparation of all solutions throughout this study. Fabrication of TNTs on Ti Wires. Ti wires were annealed at 500 °C for 2 h. Annealed wires were sonicated in acetone and dried in N2 prior to the polishing step. Electropolishing was performed in perchloric acid electrolyte, with butanol and ethanol (in the ratio of P/B/E = 1:6:9) at 25 V, using a special electrochemical setup maintained at 4 °C. Later the polished wires were cleaned with deionized (DI) water and sonicated in acetone/ethanol. Electrochemical anodization of the Ti wires was carried out by exposing a specific length (1 cm) of the Ti wire (via masking) to the ethylene glycol electrolyte (with 1% v/v water and 0.3% w/v NH4F) at 75 V for 20 min maintained at 25 °C. Chitosan Modification of TNT/Ti Wire. A solution of chitosan [1% (w/v) chitosan +0.8% (v/v) acetic acid in deionized water] was prepared. Chitosan was applied to the TNT/Ti wire using a dipcoating process, which was performed by dipping the TNT/Ti wire into the polymer solution, followed by vertical hanging in air at 25 °C for 30 min. The wires between consecutive dipping were dried. The thickness of the deposited polymer membrane was controlled by the number of dipping steps. In this study, usually three dipping steps were performed to obtain an optimized film thickness. Later, single chitosan coated TNT/Ti wire implant was immersed in 20 mL PBS (pH 7.4). At predetermined time intervals the samples were removed from the PBS and dried at room temperature (25 °C), followed by preparation for imaging. The study was performed in triplicates for each time point. Various parameters including substrate topography, immersion solution and pH, time of immersion and thickness of chitosan were also varied to study their influence on chitosan film transformation. To compare the precipitation rates, coating was also performed on various substrates, including rough Ti, electropolished smooth Ti, glass, and Si wafers. Besides PBS (pH 7.4), water and various pH solutions of PBS (pH 3.0 and 5.0) were also used as immersion solutions. Furthermore, in separate experiment, the thickness of the chitosan layer on TNTs (controlled by number of dip coating steps) was also varied, prior to immersion in PBS. Synthesis of Iron Oxide Nanoparticles and Incorporation in Chitosan Coating. Iron oxide nanoparticles (NPs) were prepared by hydrothermal process at 160 °C/5 h following a procedure described elsewhere.25 To incorporate NPs in the chitosan coating, 70 mg of NPs were dissolved in 5 mL of chitosan solution. TNT implants were coated with chitosan three times, with one of the three layers having NPs: Ch-1 + NPs (only first layer with NPs) and Ch-3 + NPs (only third layer with NPs). The other coatings were performed with chitosan solution only (without any NPs present). Implants of each 1262

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Figure 1. SEM images showing titania nanotubes (TNTs) on Ti wires, chitosan modification and transformation of chitosan into microtubes (CMTs): (a−c) TNT arrays on Ti wire, (d, e) top and cross-sectional view of chitosan-coated TNTs prior to immersion in PBS, (f, g) top-view of the TNT/Ti wire with CMTs, and (h) EDXS analysis confirming that CMTs were primarily composed of chitosan (with impurities from PBS buffer and TiO2 signal from the nanotubes). TNT/Ti wires (anodized at 75 V for 20 min) dip-coated in chitosan solution (three times) were immersed in PBS (pH 7.4) for 3 weeks. type were prepared in triplicate, and separately immersed in 20 mL of PBS (pH 7.4) and characterized at various time intervals. Structural Characterization. Structural features of the prepared TNT-Ti implants before/after chitosan coatings, after PBS immersion and for other treatments, were characterized using a field emission Scanning Electron Microscopy or SEM (FEI Quanta 450). Please note that the chitosan-modified implants (before/after immersion in various media) were dried in air at 25 °C prior to preparation for imaging, and after imaging, the samples were discarded. The samples were mounted on a holder with double-sided conductive tape and coated with a layer of platinum 5 nm thick. Images with a range of scan sizes at normal incidence and at a 30° angle were acquired from the top and bottom surfaces as well as cross sections. EDXS (energy dispersive X-ray spectroscopy) was also performed to reveal the elemental composition of various implant modifications.

3. RESULTS AND DISCUSSION Formation of Chitosan Microtubes (CMTs) on Titania Nanotube (TNT) Implants. Typical structure of TNT/Ti wire substrates prepared by electrochemical anodization (EA) of Ti wire is presented in Figure 1a,b. Under anodization conditions used in this study, TNT arrays are formed with an average tube diameter and length of 50 ± 4.2 nm and 10 ± 0.5 μm respectively, although the dimensions of the TNTs can easily be tailored using various anodization parameters.2 Despite the presence of microscale cracks in the TNT layer, as seen in Figure 1b, which arise due to the volume expansion of the anodic film, the TNT structure is mechanically stable and well-adherent onto the underlying substrate.13 Furthermore, the EA approach used to fabricate nanotubes represents a simple, cost-effective, and scalable technology, which allows 1263

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Biomacromolecules easy tailoring of TNT features, such as dimensions, geometry, and chemistry.26 To modify the implants, dip-coating of the TNT/Ti wires in the chitosan solution was performed, which represents a very simple and cost-effective approach. The motivation behind using chitosan to modify TNT implants was to promote bone cell functions and aid in controlling release of therapeutics from TNTs, as reported previously.17 Furthermore, inherently antibacterial and investigated for various bone tissue engineering applications, chitosan has been recognized as a promising bone implant modification strategy. SEM images of the topview and cross-section of the 3× dip-coated TNT implants is shown in Figure 1d,e. The number of dip-coats decides the thickness of the polymer coat, and for the 3× coating, a thickness of 380 ± 15.24 nm was obtained. The images also show that open-pores of the TNTs were totally covered with the chitosan film, which appears to uniformly cover the entire area of the implant. We have also shown previously that varied thickness of chitosan coating on TNTs (achieved by varying dip coating numbers) can decide the release kinetics of the drugs incorporated inside the nanotubes.5,6,17,18 To explore the structural transformation of chitosan film in the presence of physiological fluids, mimicking the conditions post-implantation, the implants were immersed in PBS solution (pH 7.4) in vitro. PBS represents an isotonic fluid that matches the osmolarity and ionic concentration of body fluids such as blood. TNT implants were immersed in PBS for 3 weeks, followed by continuous imaging of changes on chitosan film using SEM. The SEM images of chitosan film after 3 weeks of immersion in PBS are presented in Figure 1f,g and clearly show the appearance of a randomly arranged mesh of microtubes covering the entire surface of the TNT/Ti wire implants. These images also show that the chitosan coating remained underneath these microtubes, which indicates that after 3 weeks immersion, the chitosan coating is only partially consumed to form these structures. The study was repeated many times and structural transformation of chitosan film into chitosan microtubes is confirmed reproducibly in all cases. These formed CMTs were found to have an average thickness of 1.42 ± 0.24 μm. The EDXS analysis of these structures revealed the presence of C and N, along with TiO2 (from TNTs) and other salts from PBS in the structure, which implies that the tubes are primarily composed of chitosan (Figure 1h). Similar studies were also performed on TNTs fabricated on flat Ti foil, and formation of CMTs was observed (data not presented). To further characterize the formed chitosan microtubular structures a series of high-resolution SEM images were collected and presented in Figure 2. Microtubes formed into a mesh like mat covering the entire surface of the implant, with some areas having very dense coverage (Figure 2a), and other areas were covered with lower density (Figure 2b). In Figure 2b,c, clear evidence of the underlying chitosan coating can be seen, along with the presence of cracks in the anodic film. The microtubes appear to bridge over small cracks, which break up the smooth chitosan underlayer, which indicates that CMTs are formed from the chitosan film. In Figure 2c, a close-up of the microtubes shows that they appear to be comprised of characteristic periodic segments. In order to visualize the cross-section of the microtubes, the sample was mechanically fractured and characterized imaged SEM (Figure 2d,f). These images clearly confirm hollow structure of the microtubes, which is further confirmed by the “deflated” state of some parts of tubes (Figure 2e). These results show that in situ

Figure 2. SEM images showing the surface features of chitosan microtubes (CMTs) on chitosan coated TNT/Ti wires (a−f). Chitosan solution was dip-coated onto TNT/Ti wire (3×), followed by immersion into PBS (pH 7.4) for 3 weeks. Images (d, f) show chitosan-coated TNT samples that were mechanically fractured to visualize the cross-section of the formed chitosan microtubes.

precipitation of coated chitosan film on implant substrates can create a unique microscale tubular mesh on already bioactive TNTs, which is expected to upregulate bone cell functions.27 Please note that structural transformation of chitosan coating on TNT implants does not compromise the release of drugs from TNTs, which will continue as the coatings are consumed/transformed into microtubular structure. Briefly, as the chitosan layer is transformed, surrounding buffer is expected to enable release of drugs from TNTs, driven by the diffusion gradient. Role of Underlying Substrate Topography. To explore the role of underlying substrate topography on transformation of chitosan film and the formation of CMTs, several surfaces were studied, including unpolished Ti (∼microrough), electropolished Ti (smooth), glass, and silicon wafers. The chitosan films on these samples were prepared by the same dip-coating conditions, later followed by immersion in PBS (pH 7.4) for a period of 3 weeks. SEM images showing the top surface of these control samples are presented in Figure 3. Clearly the chitosan film (Ch-film) dissolves/transforms in a different manner on these surfaces, with none showing any signs of the formation CMTs or similar structures. For Ti (microrough surface), there exists signs of swelling and breaking of the Chfilm but no visible signs of microtube formation (Figure 3a). For perfectly smooth electropolished Ti surface, polymer swelling can be seen (Figure 3b). On the other hand, for glass and Si, the Ch-film demonstrated signs of swelling and 1264

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The comparative SEM images of chitosan films on TNTs after 5 week immersion in PBS (pH 7.4) and water (deionized, DI) are presented in Figure 4a,b. In contrast with immersion in

Figure 3. Top-view SEM images showing the influence of surface topography of the underlying substrate on the transformation of chitosan coating in the presence of PBS: (a) rough Ti, (b) electropolished smooth Ti, (c) glass, and (d) silicon wafer. Chitosan was dip-coated three times on each substrate, followed by immersion in PBS (pH 7.4) for 3 weeks. Figure 4. Top-view SEM images showing the fate of chitosan coating performed on TNT/Ti wire implants, post-immersion in various solutions: (a) PBS pH 7.4 (5 weeks), (b) deionized water (5 weeks), (c) PBS pH 3.0, 1 week, and (d) PBS pH 5.0, 1 week. Please note that for (c, d) a one-week time-point is presented as the chitosan coating was completely dissolved due to low pH of the immersion solution.

crystal formation (Figure 3c,d). From this study it can be concluded that, for the formation of CMTs, the presence of a nanorough surface with TNTs is important, whereas for microrough and smooth surfaces, the polymer swells without any microtube formation. XRD analysis of the chitosan layer on TNTs and bare microrough Ti, before and after immersion in PBS is also performed, and the results are presented in Figure S1 (Supporting Information). The presence of TNTs can influence chitosan precipitation/transformation into CMTs in two ways: template-effect and presence of anodization electrolyte. While CMTs formed only in the presence of TNTs, in-depth analysis of the role of TNTs in assisting formation of CMTs is out of scope for this paper, which is more focused on defining optimized conditions that contribute toward formation of these novel structures. Furthermore, other properties of TNTs related to their chemical composition can be considered to have impact on structural transformation of chitosan film, as CMTs did not form on control materials. The anodization process to prepare TNTs film requires electrolyte which contains ethylene glycol and [TiF6]− complexes. Small quantities of these compounds get adsorbed on/inside the porous TNTs surface during the fabrication process, and complete removal of such impurities is very challenging. The electrolyte (pH 7.8−8.0) entrapped inside nanotubes and microscale cracks, can be expected to come into direct contact with the chitosan film. Furthermore, its presence on the surface and possible release after immersion of the implant in PBS solution could impact the surface energy of TNTs, and thereby could influence the precipitation/ transformation rate of chitosan film which could potentially contribute to the formation of CMTs. Results presented in Figure S2 (Supporting Information) support this claim. However, it was difficult to prove this hypothesis and in the following sections we explored some other parameters to propose the mechanism behind CMT formation, which is discussed in the last section. Influence of Immersion Solution and pH. We investigated the effect of the type of immersion solution and pH on CMT formation, using water as an alternative solution.

PBS, which causes most of the chitosan film to partially dissolve and get transformed into CMTs (Figure 4a), immersion in water did not yield CMTs (Figure 4b). Instead, the majority of the chitosan coating dissolved, leaving very thin chitosan film (with the impression of the underlying TNTs). Please note that the study involving immersion in water and PBS (pH 7.4) was monitored weekly via SEM imaging (data not presented); however, since CMTs formation was not seen in the presence of water, the study was continued until 5 weeks. It is wellknown that chitosan solutions (which are acidic) are mostly coated as films by evaporation of water. Since these films already contain acid (in this study acetic acid), they are readily soluble in water with specific dissolution rates.28 The presence of acidic groups in the polymer film creates a slightly acidic local environment in the vicinity of the coating and can facilitate polymer dissolution. This can possibly explain why the chitosan film on TNTs dissolves faster in DI water, in comparison with PBS (pH 7.4). This can also be attributed to both the absence of ionic salts and the slight pH differences between DI water and PBS. To investigate the effect of pH of the immersion buffer solution on CMT formation, the study was extended to solutions of PBS with varying pH. We considered various pH values relevant to different body organs/tissues: blood (7.4), mouth (6.5−7.5), stomach (1.5−3), and intestine (5−7). Three pH values (3.0, 5.0, and 7.4) were analyzed with respect to the formation of CMTs. The implants were examined every week (using SEM imaging); however, the most significant results, in this case, only week 1, whereby for low pH solutions (pH 3.0 and 5.0), very high dissolution (acid hydrolysis) (with no signs of CMTs) was seen, is presented in Figure 4c,d. Clearly, different dissolution rates of chitosan films can be seen, corresponding to each pH value. For both buffer solutions (pH 3.0 and 5.0), the dissolution of chitosan was accelerated, 1265

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Biomacromolecules showing complete disintegration of the chitosan film and exposure of the underlying TNTs surfaces. SEM images revealed (Figure 4c,d) that the film was nearly dissolved, exposing the underlying TNTs surface and cracks. These results are supported by other studies showing enhanced chemical dissolution rates of chitosan at lower pH.29 Comparing the chitosan film between PBS pH 7.4 and 3.0/5.0, the CMTs formation starts in around 2−3 weeks for pH 7.4 (results presented later); however, due to the acidic environment in the case of pH 3.0/5.0, the bulk of the chitosan coating dissolved very soon in/under 1 week. The dependence of the CMTs formation in the presence of PBS with different pH can be attributed to a combination of pH and presence of ionic species.29,30 The pH of the solution and, hence, the selective dissolution of chitosan appears to be critical determinants of the formation of CMTs. Clearly chitosan tends to dissolve under acidic conditions due to protonation of amine groups; however, for pH > 7, some chains remain susceptible to dissolution, especially shorter polymeric chains with a higher degree of deacetylation (more amine groups). At higher pH, a soluble−insoluble transition allows for the formation of interpolymer associations, by reducing chitosan’s electrostatic repulsions, and providing conditions for the formation of fibers, films, or hydrogels.31,32 Furthermore, previous studies have shown that neutralization of the chitosan amino groups by anions present in the PBS could lead to removal of interchain electrostatic forces and allows for extensive H-bonding and hydrophobic interactions between chains.33 This induced gelation or precipitation can be avoided by maintaining the temperature between 4 °C, and 15 °C, but in the current experimental procedures, the temperature was 25 °C, which allowed for substantial ionic neutralization or gelation.33 Hence, combining appropriate pH and ionic species, facilitates selective dissolution of more soluble chains, and the simultaneous precipitation/neutralization, resulting in less soluble gelled chains, leading to formation of microtubes. The detailed mechanism behind CMTs formation is discussed in the last section, taking into account all the contributing factors. Influence of the Thickness of the Chitosan Layer on the Formation of Chitosan Microtubes (CMTs). To elucidate the effect of the film thickness and number of coatings on the formation of CMTs, two different strategies were employed, as presented in Scheme 2. First, to understand the role of the number of coats, TNT wire samples with varied numbers of chitosan dip coatings were prepared, including one, two, and three dippings (Scheme 2a). In the second strategy, magnetic nanoparticles (Fe3O4) were incorporated in the chitosan solution prior to dip-coating on TNTs. This experiment was designed so that only first chitosan film (Ch1) or third chitosan film (Ch-3) contains nanoparticles (NPs), as represented in Scheme 2b. The remainder of the chitosan coats were dip-coated from chitosan solutions that did not contain NPs. The rationale for these experiments was to determine the role of chitosan coat thickness in the formation of microtubes and how the layers dissolve in PBS with time. After immersion in PBS for 3 weeks, the surface features of the chitosan film on TNTs were examined using SEM and results are presented in Figure 5. The SEM images clearly indicate that the formation of CMTs is dependent on the thickness of chitosan film. Thin chitosan film prepared by single step dip-coating (1× chitosan) dissolved almost completely within 3 weeks, as evident from the top-view and crosssectional SEM images showing underlying pores of the TNT

Scheme 2. Scheme Showing the Experimental Setup for Investigating the Role of the Chitosan Film Thickness on the Transformation and Self-Ordering Process on TNT Surfaces Immersed in PBS Solutiona

a

(a) Varied number of chitosan-coating layers on TNT surfaces (controlled by number of dip-coating steps) and (b) incorporation of nanoparticles (NPs) inside the chitosan layers.

Figure 5. Influence of chitosan film thickness (number of layers on TNTs, controlled by number of dip-coating process) on the formation of chitosan microtubes (CMTs; based on schematic shown in Scheme 2a). SEM images showing high-magnification top and cross-sectional view of chitosan film (prepared by 1, 2, or 3 dip-coats on the TNT/Ti surface) after 3 weeks of immersion in PBS (pH 7.4): (a−c) single coat [(b) (inset) before immersion in PBS], (d, e) double coat, and (f, g) triple coat of chitosan.

layer (Figure 5a and 5c). The image presented in Figure 5b (inset) shows the TNT surface with single chitosan coating 1266

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Biomacromolecules prior to immersion inside the PBS buffer, which implies that a very thin coating is present (∼130 nm). The formation of CMTs is not observed on this chitosan film and there are several reasons to explain these results. The first is that very thin chitosan film from single coating is very firmly adhered onto the nanotube structures (∼50 nm diameters) with partial depressions inside nanotubes. The second is that dissolution rate of thin chitosan film is faster compared with thick film. Therefore, the prospect for the formation of microtubes by curling or delamination process is significantly reduced. In case of two steps dip coated chitosan films (2× chitosan), CMT formation was observed with a very thin coating of chitosan underneath (Figure 5d,e). This result indicates that certain thickness of chitosan film (∼250 nm) is required for the formation of CMT structures. Finally, for three-step-coated chitosan film (3× chitosan), CMTs formation on TNTs surface was clearly observed, with a thicker coat of chitosan underneath (Figure 5f,g). These results show that the chitosan precipitation, dissolution and microtube formation are greatly influenced by the number of coatings or the thickness of the chitosan film. The possible explanation for these observations is that multiple coats are required to give a layer of sufficient thickness to form a CMT structures. As explained previously thin chitosan layer is likely to be well adherent to the TNTs structures, hence making the formation of wrinkles on the films very difficult (explained in details later). On the contrary, for a thicker layer, the upper layers of the polymer film would be able to contract and form wrinkles. Availability of substantial material layered further away from the TNTs (in the case of thicker layers) can also facilitate easy movement of molecular chains, which seems to be important for formation of CMT. For the second experiment, magnetic nanoparticles (NPs) were incorporated into the different layers of chitosan coating so as to visually ascertain the contribution of each layer on the formation of CMTs (Scheme 2b). Please note that, for this experiment, a total of three dip-coating steps were performed, with the exception of including NPs in the first coat (Ch-1 + NPs) and the third coat (Ch-3 + NPs) of chitosan. The remaining two coats were made using the normal chitosan solution (without any NPs). This permitted a polymeric coating on TNTs with three distinct layers. To further simplify, Ch-1 + NPs sample has Ch-2 and Ch-3 without any NPs, and Ch-3 + NPs sample has Ch-1 and Ch-2 without any NPs. The intermixing of NPs among different layers/coats was avoided by complete drying between dip-coats, prior to adding the next layer. The results as presented in Figure 6 shows SEM images of these chitosan films after immersion in PBS (pH 7.4) for 3 weeks. Week 1 results for both the samples show that the first layer has started to swell/dissolve. The top view of the Ch-1 + NPs sample shows no sign of microtubes (due to the early timepoint) and also no impressions of the underlying NP agglomerates, which can be assumed to be buried in the first coating (Figure 6a). For the Ch-3 + NP sample, agglomerates of the NPs are observed, and for some areas the NPs are exposed to the PBS (Figure 6b). This signifies that Ch-3 layer has started to dissolve, revealing the embedded NPs. Following 3 weeks of PBS immersion, microtube formation is evident in both samples (Figure 6c,d), with the main difference being that NPs are present in the same layer as the CMTs in the Ch-3 + NPs sample, whereas the NPs appear to lie below the CMTs in the Ch-1 + NP sample. From this result, it may be concluded that microtube formation is initiated in the top surface that is

Figure 6. Investigating how each chitosan layer on TNTs transforms and contribute toward formation of chitosan microtubes (CMTs; based on schematic shown in Scheme 2b). Top-view SEM images showing chitosan modified TNTs after immersion in PBS for 1 and 3 weeks: (a, c) Ch-1 + NPs (only the first coat contained nanoparticles/ NPs), and (b, d) Ch-3 + NPs (only the third coat contained NPs).

exposed to the PBS solution. The delayed exposure of NPs on Ch-1 + NPs (after 3 weeks, Figure 6c) is the evidence that protonated chitosan molecules are consumed systematically from the top coat (Ch-3) to the bottom (Ch-1), to form a network of CMTs with time. It can be also concluded that the layered films of chitosan are effectively converted to CMT and eventually expose the pores of the TNT. The thickness of these layers can be used as one of the parameters to delay the exposure of the TNT for effective drug release. Furthermore, the incorporation of magnetic NPs does not interfere with the ability of the chitosan film on TNTs to form microtubes. The implication of these findings is that therapeutic or other functionalities involving magnetic NPs could be integrated into chitosan-modified implants, so that as the film disintegrates into microscale tubes, the substance would be released. To confirm the nature of each feature identified in Figure 6, EDXS analysis of the CMTs and NPs was performed. The results for Ch-1 + NPs sample after 5 weeks of immersion in PBS pH 7.4 are presented in Figure 7. In the SEM image (Figure 7a), three distinct areas are marked, which were characterized by EDXS. Area-1 features the underlying TNT substrate, which is confirmed to contain TiO2, with some signs of NPs. CMTs are present in area-2, which contains elements that are attributed to the chitosan (along with some buffer salts). For area-3, an agglomerate of NPs is clearly present, as confirmed by the high percentage of iron oxide. Time-Course of Chitosan Microtubes (CMTs) Formation. In order to better understand the formation of CMT structure, the time-course of precipitation/neutralization and structural transformation of chitosan film (three steps dipcoated onto TNT/Ti wires) was studied. This was achieved by immersing TNTs coated with chitosan films in PBS (pH 7.4) for various predetermined time intervals ranging from 1 day to over 5 weeks, followed by SEM imaging. Before immersion in the PBS, top-view images confirms the uniform coating of chitosan film covering the TNTs surface (Figure 8a). After 1 day of immersion in PBS (Figure 8b), the coating remained 1267

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Figure 7. Identifying the chemical composition of chitosan products during precipitation/neutralization of chitosan film (incorporating nanoparticles/NPs) and microtube formation process. Only the first coat of chitosan on TNTs contained NPs (Ch-1 + NPs), and the immersion in PBS pH 7.4 was continued for 5 weeks. (a) Top-view SEM image, and (b−d) EDXS analysis of three distinct areas identified in the SEM image: (b) TNTs, (c) CMTs on TNTs, and (d) NP agglomerates.

intact, but appeared to wrinkle. The appearance of wrinkles or creasing of the biopolymer is likely due to localized relief of stress induced by precipitation/neutralization, whereby conformational changes occur permitting the NH2 groups to form intermolecular H-bonds, thereby aiding in the formation of the precipitate.34 Following 1 week of PBS immersion (Figure 8c,d), further wrinkling of the film occurred, with signs of the coating transforming into tube-like structures. Upon contact with PBS, neutralization/precipitation caused the lower solubility chitosan chains (longer/less amine groups) to gel, yielding localized shrinkage and wrinkling. The gelation can further make the polymeric chains to change conformation, that is, bunch up, excluding water and, hence, impeding the dissolution.34,35 As shown in earlier results, at lower pH, PBS causes the entire polymer to dissolve; as the pH is low enough to dissolve even the gelled polymer (more protonation of amine groups facilitates dissolution). When the coated implants were immersed for more than 3 weeks, clear transformation of the polymer film into a tubular structures was observed, as shown in Figure 8e−g. The tubes can be seen disintegrating from the bulk film structure (Figure 8e), which indicates that the chitosan coating is composed of regions of varied solubility, whereby the soluble chains are consumed initially, leaving a less-soluble polymer. Another feature, evident after week 3 immersion in PBS, was the presence of underlying chitosan film, which still covered the TNTs. Finally, after 5 weeks immersion, the underlying TNTs were revealed with some signs of microtubes still present and thin patches of undissolved chitosan (Figure 8h,i). These images confirm that the microtubes start appearing after 2−3 weeks of immersion in PBS and, in around 5 weeks, the bulk of the chitosan is dissolved, with some fragmented coating and a few microtubes remaining. For clarity, the “bulk chitosan film” is referred to as the coating underneath the “microtubes”. Elucidating the Mechanism of Microtube Formation on TNTs. From presented results, we hypothesize the possible

Figure 8. Influence of time of immersion on the transformation of chitosan coating (dip-coated thrice) on TNT implants into microtubes. SEM images showing the top view of implants for various immersion times in PBS (pH 7.4): (a) before immersion in PBS, after immersion for (b) 1 day, (c, d) 1 week, (e−g) 3 weeks, and (h, i) 5 weeks.

mechanism of the formation of CMTs from chitosan films on TNT surfaces. The mechanism can be summarized in the following steps: (a) Chitosan film swelling and wrinkling: It has been reported that chitosan films initially swell when exposed to PBS (pH 7.4) which is followed by reduction in volume over time.29 Research has also indicated the possibility of cross-linkage between chitosan’s cationic amino groups and PBS’s phosphate anions.36−38 Initially chitosan film absorbs water/swell, while simultaneously, chitosan’s amine groups get protonated. This phenomenon can create surface defects to minimize energy and thereby the surface of chitosan wrinkles, creating microscale peaks and valleys (Scheme 3a). (b) Variation in pH and insolubilization of chitosan: Microenvironment at the PBS/Chitosan interface. Since the local environment near chitosan film is expected to be slightly acidic, upon immersion in PBS (pH 7.4), a sudden change in pH occurs.39 Variation in pH can significantly change the charge state/properties of chitosan (due to presence of amino groups). Further1268

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Scheme 3. Schematics Showing the Proposed Mechanism of Wrinkling, Precipitation, and Formation of Chitosan Microtubes when Chitosan-Coated TNT/Ti Wire are Immersed in PBS (pH 7.4) for Extended Periods of Timea

a (a) Wrinkling and insolubilization of chitosan due to sudden change in pH, (b) neutralization of chitosan by PBS buffer (formation of semipermeable membrane), (c) formation of microtubes due to surface energy and interpolymeric associations (due to continued neutralization), and (d) SEM images presenting different transformation stages of chitosan microtube formation, corresponding to the numbers of the schematic in (c).

The role of the PBS solution is first highlighted based on our results showing that CMTs are not formed in the presence of water. In the phosphate buffer solution, many ionic species are present, which substantially influence the reaction kinetics of chitosan dissolution. For the presence of multiple ions, there is a possibility of “ion-induced in situ precipitation” or “neutralization”, which has been reported elsewhere, especially in the presence of alkaline medium.43−47 When polycationic NH3+ encounter negatively charged species such as phosphate ions, NH3+ converts to NH2 following the equation

more, it is well-known that when pH > 6, chitosan loses charge and becomes insoluble (deprotonation of amines).40 Microenvironment at the chitosan/TNTs interface: Titania nanotubes (TNTs), fabricated using anodization, contains electrolyte, which often gets included deep into the structures, and its removal can be very challenging. Furthermore, TNTs on Ti wire results in the presence of cracks in the anodic film, which can extend to the length of the nanotubes.13 As a result, a high amount of electrolyte can be assumed to be present at the interface between chitosan coating and TNTs. The nano/ microrough surface promotes contouring of chitosan film and the contact with organic electrolyte (pH ∼ 8.0), assists in wrinkling/curling, and generates a poorly soluble region.40,41 (c) Continuous phosphate ion penetration and interpolymeric associations: At high pH (>6.5), chitosan amines become deprotonated and reactive (reducing chitosan’s electrostatic repulsions), which can facilitate various interpolymeric associations that can cause fiber or network formations.42 This further results in removal of repulsive interchain electrostatic forces and enables Hbonding and hydrophobic interactions between chitosan chains. This coupled with maintaining minimum surface energy on already wrinkled microrough (peaks and valleys) and insoluble chitosan results in formation of CMTs. The process of PBS-induced precipitation/ interpolymeric associations is discussed in detail in the following sections.

Ch‐NH3+ + HPO4 2 − → Ch‐NH 2 + H 2PO4 −

Ch-NH2 is sparingly soluble or insoluble as compared to the cationic Ch-NH3+, and this immediately results in the generation of an insoluble semipermeable membranous (SPM) structure (Scheme 3b). This initial Ch-NH2 SPM, which forms when cationic chitosan comes in contact with negative ionic species, allows ions such as Na+, K +, OH−, Cl−, PO43−, and so on, to permeate based on charge equilibrium; however, the chitosan still remains largely impermeable.44 This in-flow of ions from the PBS medium into the SPM created by Ch-NH2 continues and results in the formation of more SPMs composed of insoluble Ch-NH2. This process will continue until the chitosan or the active negative ions are consumed. Insoluble Ch-NH2 forms microtubular structures and not just layer-after-layer composed of insoluble SPMs, because at the end of 5 weeks of immersion, CMTs are present on TNTs, rather than the entire coating being converted into the insoluble Ch-NH2 form (Figure 4a). This can be attributed to surface energy effects, which are enhanced for precipitated 1269

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of 7.4 (compromised solubility). We propose that selfformation of unique chitosan microscale tubular structures on nanotubular TNTs can open new possibilities for in situ synthesis strategy for optimizing implant performance. Furthermore, the properties and applications of multifunctional biopolymers such as chitosan has been explored with significant advancements over the last few decades; however, the fate of such biopolymer modifications on the nanoengineered surfaces has not been researched. This is crucial to further advance the properties/applicability of established materials (chitosan and titania nanotubes in the current study) to explore their combination into one device.

and curled/swollen chitosan coating, and the presence of interand intramolecular forces. As a result of polymeric chain rearrangements or a change in conformation, surface defects occur, with local delamination of the chitosan film, resulting in creasing or folding (Scheme 3a). This curled region, which on reaction with negative ions converts to insoluble Ch-NH2, and this process of conversion of Ch-NH3+ to Ch-NH2 continues, and the surrounding chitosan is also consumed into this process and contributes to the microtube formation (Scheme 3b,c). This is also evident from SEM images, which show that the microtubes arise from the bulk of the chitosan coat, especially in Scheme 3d(2), where the chitosan coat can be seen still bound to the microtube. Furthermore, it has been established that solubility of chitosan depends on the nature and the pH of the solvent, where the presence of salt can significantly influence the conformational behavior of chitosan.33−35 This phenomenon can also be related to Gibbs-Donnan equilibrium, whereby the imbalance between distributions of ions (like Na+, Cl−, PO43−, and so on, from PBS) across the polymeric membrane induces electrical imbalance, which can lead to high surface compressive forces to minimize surface energy. It is important to note that the chitosan layer is exposed to two regions of varied pH, at PBS and TNT interfaces, and both can contribute toward the generation of insoluble/sparingly soluble chitosan. Previous studies have shown that the presence of organic solvents (anodization electrolyte contains ethylene glycol) can cause poor solubility of chitosan.41 This effect can also be enhanced with the presence of [TiF6]− complexes in the anodization electrolyte; however, in-depth analysis of the relationship between [TiF6]− and chitosan is required to arrive at a conclusion, which will be reported in future studies. Additional information regarding the impact of anodization electrolyte is presented in the Supporting Information (Figure S2). Another phenomenon of thickening of Ch-NH2 SPM can explain why hollow tubular structures are obtained and not solid tubes. Over time, as the Ch-NH2 structures are formed layer-by-layer, the SPM starts to thicken, as represented in Scheme 3c. This ultimately leads to a time-point where further permeability of the ionic species across the diffusion gradient is compromised.47 This in turn leads to complete “switch-off” of the SPMs’ permeability, preventing further penetration of ions to convert more of cationic chitosan into the insoluble form. A tubular shell can be envisaged, with a core-like structure, whereby the outer shell is composed of multilayers of insoluble Ch-NH2 SPMs compressed together and a core of soluble cationic chitosan. This overtime results in dissolution of the “core” material, resulting in a hollow tube-like structure composed of insoluble Ch-NH2. Also inter- and intralayer hydrogen bonding can contribute toward further-curling of the soluble core composed of Ch-NH3+, leading to tubular morphology. Discerning the exact mechanism whereby chitosan coated onto a nanoporous substrate (TNTs on Ti wire), immersed in PBS buffer (at pH 7.4), precipitates into microscale tubes (CMTs), is difficult. However, for the presence of multiple parameters including TNTs (anodization electrolyte and template-effect), pH, and phosphate buffer, means that the mechanism cannot be attributed to a single effect. The possible contributing factors, based on the optimizations performed, can be summarized as TNTs nanoporous substrate (anodization electrolyte/surface energy effects), presence of salt-rich aqueous solution (aqueous dissolution, ionic neutralization), and a pH

4. CONCLUSIONS The in situ formation of chitosan microtubular structures (CMTs) on the surface of titania nanotubes (TNTs) wire implant is reported. The structural transformation of chitosan films into microtubes after immersion in PBS solution with a very unique morphology is demonstrated for the first time. Selfgeneration of dual topography with microscale tubes (CMTs) on nanotube modified implants, was obtained using in situ process and mimicking the microenvironment in the vicinity of such implants inside the target tissue in patient’s body. The various parameters that influence the formation of these microtubes including substrate topography, type of immersion solution, pH, thickness of chitosan film, and the time of immersion, were studied to elucidate the mechanism of CMT formation. The results revealed that formation of CMTs on TNT/Ti wire is dependent on surface topography (nanotubular), pH (7.4 optimal), thickness of film (>250 nm), and the time of immersion (>3 weeks). Based on this information, a mechanism of microtube formation based on in situ precipitation, upon reaction of active ionic species present in the PBS buffer at pH > 7 with the cationic chitosan was proposed. The presented in situ formation of chitosan microtubes on TNT coatings of implants provides new understanding of the transformation of bioactive polymers used as therapeutic or osseointegrating implant modifications. The knowledge of generating unique micro/nanorough topography, in combination with future studies aimed at performing cellular studies, could further aid in designing the next generation of improved bone implants.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b01037. XRD analysis of chitosan-modified TNT and Ti implants immersed in PBS, and influence of anodization electrolyte, drying procedure, and preparation steps for SEM imaging on chitosan transformation into CMTs on TNTs when immersed in PBS (PDF).

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AUTHOR INFORMATION

Corresponding Author

*Phone: +61 8 8013 4648. E-mail: [email protected]. au. Author Contributions ‡

These authors contributed equally (L.J. and R.K.).

Notes

The authors declare no competing financial interest. 1270

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ACKNOWLEDGMENTS The authors acknowledge the financial support of ARC DP 120101680, FT 110100711, and The University of Adelaide. Also acknowledged is the characterization support from Adelaide Microscopy, The University of Adelaide.

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ABBREVIATIONS TNTs, titania nanotubes; PBS, phosphate buffer solution; CMTs, chitosan microtubes; Ch-film, chitosan film; NPs, nanoparticles; SEM, scanning electron microscopy; EDXS, energy dispersive X-ray spectroscopy

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