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Chitosan–nanobioactive glass electrophoretic coatings with bone regenerative
Chitosan–nanobioactive glass electrophoretic coatings with bone regenerative
Chitosan–nanobioactive glass electrophoretic coatings with bone regenerative
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Chitosan–nanobioactive glass electrophoretic coatings with bone regenerative

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Chitosan–nanobioactive glass electrophoretic coatings with bone regenerative

  1. 1. View Article Online / Journal Homepage / Table of Contents for this issue Journal of Dynamic Article Links < C Materials Chemistry Cite this: J. Mater. Chem., 2012, 22, 24945 www.rsc.org/materials PAPER Chitosan–nanobioactive glass electrophoretic coatings with bone regenerative and drug delivering potential Kapil D. Patel,ab Ahmed El-Fiqi,ab Hye-Young Lee,ab Rajendra K. Singh,ab Dong-Ae Kim,abc Hae-Hyoung Leeac and Hae-Won Kim*abcPublished on 03 October 2012 on http://pubs.rsc.org | doi:10.1039/C2JM33830K Received 14th June 2012, Accepted 3rd October 2012 DOI: 10.1039/c2jm33830k Nanocomposites with bone-bioactivity and drug eluting capacity are considered as potentially valuable Downloaded by Dankook University on 23 November 2012 coating materials for metallic bone implants. Here, we developed composite coatings of chitosan (CH)– bioactive glass nanoparticles (BGn) via cathodic electrophoretic deposition (EPD). BGn 50–100 nm in size with aminated surface were suspended with CH molecules at different ratios (5–20 wt% BGn) in aqueous medium, and EPD was performed. Uniform coatings with thicknesses of a few to tens of micrometers were produced, which was controllable by the EPD parameters (voltage, pH and time). Thermogravimetric analysis revealed the quantity of BGn within the coatings that well corresponded to that initially incorporated. Apatite forming ability of the coatings, performed in simulated body fluid, was significantly improved by the addition of BGn. Degradation of the coatings increased with increasing BGn addition. Of note, the degradation profile was almost linear with time; degradation of 5–13 wt% during 1 week became 30–40 wt% after 7 weeks at almost a constant rate. The CH–BGn coatings showed favorable cell adhesion and growth, and stimulated osteogenic differentiation. Drug loading and release capacity of the CH–BGn coatings were performed using the ampicillin antibiotic as a model drug. Ampicillin, initially incorporated within the CH–BGn suspension, was eluted from the coatings continuously over 10–11 weeks, confirming long-term drug delivering capacity. Antibacterial tests also confirmed the effects of released ampicillin using agar diffusion assay against Streptococcus mutants. The CH–BGn may be potentially useful as a coating composition for metallic implants due to the excellent bone bioactivity and cell responses, as well as the capacity for long-term drug delivery. 1. Introduction process, biomimetic coating, sputtering and electrochemical treatment.4–11 Commercial pure titanium (CPTi) and its alloys have been Electrophoretic deposition (EPD) is one of the most useful and extensively used as implants in dental, cranial-maxillary facial effective coating methods available, mainly due to its simplicity reconstruction and orthopedic applications.1 This is primarily and low cost. Advantages also include the possibility of due to their excellent corrosion resistance and biocompatibility, producing a coating layer with high uniformity and variable allowing bone-implant integration.2,3 The biocompatibility of thickness (0.3–100 mm), the capacity to coat complex shapes, the metallic implants can be improved by the surface modification, ease of control over the coating composition and commercial such as the control over roughness and topography, and the availability. It is possible to apply either an anodic or cathodic coating with bioactive compositions. While the coatings are the treatment depending on the charge of the particles or molecules protective layer against corrosion of metals, they impart new being deposited.9 Using the EPD method, a range of composi- compositions to the surface, allowing a large spectrum of tions, including biopolymers,9,12,13 bioactive ceramics14,15 and possibilities of choosing compositions to trigger proper tissue composites16–21 have been deposited for biomedical implants. reactions. A number of coating techniques have been developed, Among the compositions, here we focus on biopolymer which include plasma spraying, anodic oxidation, sol–gel composites with bioactive inorganic nanoparticles. In fact, there has been significant attention to produce biopolymer composite a Institute of Tissue Regeneration Engineering (ITREN), Dankook coatings with inorganic particles by the EPD method.17–23 Inor- University, South Korea. E-mail: kimhw@dku.edu; Fax: +82 41 550 ganic particles, including hydroxyapatite (HA), carbon nano- 3085; Tel: +82 41 550 3081 tube, silica, and their combinations, introduced into the b Department of Nanobiomedical Science & WCU Research Center, Dankook University Graduate School, South Korea polymeric solutions, were enabled to form co-deposits by the c Department of Biomaterials Science, College of Dentistry, Dankook EPD process. Among the biopolymer sources, chitosan (CH) has University, South Korea been widely used, as it is biocompatible and degradable and is This journal is ª The Royal Society of Chemistry 2012 J. Mater. Chem., 2012, 22, 24945–24956 | 24945
  2. 2. View Article Online highly positively charged, allowing for the ease of cathodic EPD. 2. Results and discussion For the bioactive inorganic component, here we used novel inorganic nanoparticles, bioactive glass nanoparticles (BGn), 2.1. Properties of CH–BGn coatings which were newly developed in this study. BGn are considered to Fig. 1 shows the characteristics of the BGn prepared for the disperse well in the CH-containing acidic solution and conse- coating materials for EPD. A typical amorphous silica phase quently provide excellent bone-bioactivity to the coating layer, with only one broad peak at 2q ¼ 22.5 was noticed in the XRD thus presenting the potential for bone regeneration. pattern (Fig. 1a). The BGn were functionalized with amine CH is a natural polymer that can be obtained from the groups using APTES to allow cathodic EPD coating. While the exoskeleton of insects, crustaceans and fungi.22 It is generally FTIR spectrum of non-functionalized BGn displays bands obtained by deacetylation of its parent polymer chitin, a poly- related to the silica glass such as 544 and 1200 cmÀ1 (Si–O–Si saccharide that is widely distributed in nature. While the parent bending), 1070 cmÀ1 (Si–O–Si stretching) and 784 cmÀ1 (Si–O– chitin is insoluble in most organic solvents, CH is readily soluble Ca vibration),40,41 additional bands at 1365 and 1737 cmÀ1 in dilute acidic solutions below pH $6.0 due to the quaternisa- assigned to –NH2 stretching mode of aromatic amine alsoPublished on 03 October 2012 on http://pubs.rsc.org | doi:10.1039/C2JM33830K tion of the amine groups that have a pKa value of 6.3, which appeared after the amine-functionalization42 (Fig. 1b). The allows CH to be a water-soluble cationic polyelectrolyte.24 z-potential of the BGn measured at pH 7.4 changed from highly Because of the biocompatibility and charged property, CH has negative (À24.9 mV) to positive (+21.9 mV) after the amination, been used as biomedical materials, including scaffolds, gene confirming the successful amine-functionalization of the surface Downloaded by Dankook University on 23 November 2012 delivery systems and coating materials.21–28 Particularly for EPD (Fig. 1c). Furthermore, the z-potentials of amine-functionalized coating, CH molecules are considered effective for deposition BGn measured at pH 3–4 (the pH range of EPD solutions) under cathodic EPD conditions due to its positively charged showed much higher positive values (from +24 mV to +31 mV). nature. The TEM morphology of BGn showed the development of The BGn used in this study were sourced from a sol–gel uniform-sized particles less than a hundred nanometers (85 Æ precursor and prepared using a surfactant-mediated emulsifica- 15 nm, Fig. 1d). Prior to the EPD process, we observed the tion method. In fact, the class of BGs has long been considered properties of the BGn–CH solution. A drop of the solution one of the most potential bioactive inorganic materials in bone (10BGn–CH) was dried on a copper grid and the TEM image regeneration areas since the advent of melt-derived composi- was taken (Fig. 1e). Nanoparticles easily came close to each other tions.29–33 More recently, the nano-sized (generally tens to during the drying process, and the individual nanoparticles were hundreds of nanometers) forms of BGs such as nanofibers and separated completely enclosed by the CH matrix. The colloidal nanoparticles have been developed in anticipation of further stability of the solution was also assessed by a turbidity test. An potential applications, including nanocomposites with poly- optical transmission % of the solution was monitored every 1 h mers.34–38 The nanoparticulate form of BGs is considered to be effectively useful, being homogeneously dispersed with CH solution to preserve the colloidal status during the EPD coating process. Furthermore, the BGn in the coating layer will provide the compositional merits that can bestow excellent bone-bioac- tive and regenerative capacity. Here, we develop composite coatings composed of CH and BGn (a binary composition 85SiO2–15CaO) through the cathodic EPD technique. In fact, some recent studies on EPD coatings implemented CH composites with BG granules,15,39 where the bone-bioactive BG composition was utilized to improve the biological properties of the composites with poly- mers. Here, the application of the nanoparticulate form of BG in concert with CH for the EPD coating is thus considered a novel approach. Furthermore, the idea of providing the composite coating a capacity to deliver therapeutic molecules is believed to bring useful information on how to improve the bone regenerative potential of EPD coatings. Here we report the EPD process of CH–BGn composites, and systematically investigated the physicochemical and biological properties of the coatings, in terms of degradation, bone-bioactivity and osteoblastic cell responses. Furthermore, we sought to incor- porate drugs within the coating layer during the EPD process to improve the therapeutic potential of the coatings, which is considered to be a special merit of the EPD method. As a model drug, an antibiotic was chosen and antibacterial tests were Fig. 1 Characteristics of BGn; (a) XRD pattern, (b) FT-IR spectra carried out to ascertain the efficacy, to provide insight into the before and after amination, (c) z-potentials before and after amination, use of other bioactive molecules more relevant to bone repair and (d) TEM image, and the colloidal solution of BGn in CH; (e) TEM and regeneration. image after drying and (f) turbidity test monitored over 24 h. 24946 | J. Mater. Chem., 2012, 22, 24945–24956 This journal is ª The Royal Society of Chemistry 2012
  3. 3. View Article Online for up to 24 h (Fig. 1f). Results gave almost constant optical transmission with little fluctuation during the monitoring time, demonstrating a high colloidal stability of the composite solution. Using the aminated-BGn, we prepared colloidal suspensions in CH solution at different BGn concentrations (5, 10, 15 and 20 wt %) for the EPD process. As the EPD solution, we used 25% ethanol in water to control the electrolysis of water at high voltage, and gas evolution at the electrodes. The gas bubble formation in water solution is deleterious to the quality-control over the EPD coating layer, and the partial replacement with ethanol reduces gas evolution.9 We also observed a similar effect of ethanol, and 25% was shown to be optimal from a pilot study.Published on 03 October 2012 on http://pubs.rsc.org | doi:10.1039/C2JM33830K Within the ethanol–water mixture solution and acidic conditions (pH below 3.6), the CH molecules and BGn formed a stable colloidal state with surface z-potentials that were highly positive. Under an appropriate electrical field, those positively charged Downloaded by Dankook University on 23 November 2012 colloidal particles moved towards the cathode to be neutralized by consuming the hydroxyl groups (OHÀ) generated and consequently formed stable deposits on the cathodic substrate. During the EPD process, we observed a weight gain of the coatings by varying the deposition parameters, including pH, time and voltage. First, an acidic solution was observed to be required for the EPD; the deposition above pH 3.6 produced an inhomogeneous coating morphology. The pH values measured before and after the EPD process were observed to change very slightly (0.1–0.2). At different pH applied (pH 3.1 and 3.6), the weight of the coating (10BGn) increased with increasing voltage from 20 to 80 V (Fig. 2a). The weight gain was more pronounced as the solution became more acidic, which reflected the increasing positive nature of the CH molecules and BGn with pH decrease (as deduced from the surface electrical potential change with pH). The coating weight gain was also observed to be almost Fig. 2 Weight gains of samples during EPD coating measured at varying linear as a function of time (Fig. 2b). An observation of the coating parameters, including pH, voltage and time: (a) for 10BGn at two weight gain at different compositions (at 5 min coating time) different pHs (3.1 and 3.6) as a function of voltage, (b) for CH and revealed that the incorporation of BGn increased the coating 10BGn at 60 V and pH 3.6, as a function of time, and (c) for all weight (Fig. 2c). The weight increase as a function of the amount compositions at 5 min with varying voltage. of BGn was not linear, but appeared to be exponential. Together with the fact that the BGn addition increases the weight of composites (at a given volume), the larger coating volume (or morphology and this was more pronounced as the amount of thickness) may explain this. All the coatings produced herein BGn increased (Fig. 3b–d). The BGn appeared to be clustered, stably adhered to the metallic substrate, not allowing the ease of contrasted in brighter areas with localized sizes of around a few scratching and peeling off, and delamination even after the micrometers (larger than individual BGn). This cluster-like ultrasonic vibration in water. More in-depth tests on mechanical formation of BGn is considered to result from the EPD process properties of the coatings will be discussed in future works. as the BGn present in the CH solution are relatively stable. The At this point a possible EPD mechanism of the BGn–CH electric field applied should alter the surface electrostatic status composite is proposed. In acidic solution, CH molecules become of the BGn, possibly weakening the stability of individual positively charged by protonation, and thus easily accumulate at nanoparticles and rendering them to form cluster-like areas in the electrode by the electrophoresis.22 Moreover, the BGn, as the coating layer with sizes of a few micrometers. The literature they are also positively charged, can also deposit similarly, also reported a similar phenomenon for the clusters of nano- resulting in co-deposition with CH. In fact, the cathodic depo- particles.23,43 Strictly speaking, the BGn in the clusters should, sition of CH composites either with silica or hydroxyapatite however, be separated, surrounded by the CH molecules, (HA) has been reported elsewhere.20,21,23 In those cases, the silica moreover the clustered areas appeared to distribute at similar or HA particles are initially negatively charged, which however spatial distances throughout the coating layer – a feature not become positively charged due to the adsorption of CH mole- readily found in the composite coatings where micron-sized cules and thus co-deposit with CH. particles were initially used. However, strategies to improve the The coating morphology was observed by SEM, as shown in nanoparticle dispersion in the EPD coating layer will be required Fig. 3. While the pure CH coating showed homogenous and for further studies, and the possible ways are to provide a clean morphology (Fig. 3a), the composite coatings had a rough stronger positive charge to the nanoparticles or to decrease the This journal is ª The Royal Society of Chemistry 2012 J. Mater. Chem., 2012, 22, 24945–24956 | 24947
  4. 4. View Article OnlinePublished on 03 October 2012 on http://pubs.rsc.org | doi:10.1039/C2JM33830K Downloaded by Dankook University on 23 November 2012 Fig. 3 SEM surface morphologies of the coating layers. (a) CH, (b) 5BGn, (c) 10BGn, and (d) 15BGn. In (e), the coating layer was scratched off from the Ti substrate to reveal a coating layer (5BGn) with a level of thickness (indicated an arrow). Coatings performed at 50 V for 5 min at pH 3.6 were shown for representative examples. content of nanoparticles. The cross-section morphology of the 2.2. In vitro degradation and apatite forming ability coating layer was examined by scratching off from the Ti Some important in vitro properties of the coating layers for the substrate (Fig. 3e); a thickness of $15 mm was formed in the hard tissue applications, including degradation and bone- 5BGn coating. Similarly observed thickness was $12 mm for CH bioactivity, were also investigated. Fig. 5 shows the degradation coating, $30 mm for 10BGn coating and $48 mm for the 15BGn of the coatings with time during incubation in PBS at 37 C for coating, which corresponded well to the results of the coating periods of up to 50 days. For all coating compositions, the weight gain shown in Fig. 2c. degradation profile was almost liner with time, and the incor- The EPD coatings were further characterized, as presented in poration of BGn increased the degradation rate. For the CH Fig. 4. The exact composition of the composite coatings was coating, the degradation was $5% for 7 days, $13% for 21 days, investigated by TG analysis. For this, some parts of the coating $18% for 35 days and 34% for 50 days. For the 15BGn coating, layer scratched off from each sample were heat-treated up to the degradation was $12% for 7 days, $25% for 21 days, $32% 900 C and the weight change was monitored (Fig. 4a). The for 35 days and $42% for 35 days. The incorporation of BGn is TGA pattern of CH showed three steps in weight loss; first 22% thus considered to speed up the hydrolytic degradation of the up to $200 C was attributed to the liberation of adsorbed coatings, in the form of ionic release of the BGn and/or disso- water, and two further steps at 200–350 C and 350–600 C ciation of CH molecules. One interesting thing was the linear were from the thermal degradation of CH. Whilst CH showed release pattern observed in the coatings, which is consistent with almost 100% weight loss at $600 C, all the composite coatings the view that the coating degradation is primarily associated with preserved a certain amount of weight at the end, although the the surface erosion process. It is envisaged that the degradation three-step behavior was similarly observed. The remaining process should significantly influence the release pattern of drugs weight measured was 4.89, 9.99 and 14.84% for 5BGn, 10BGn that are incorporated within the coating layer, as discussed and 15BGn coating, respectively. The results confirmed that the subsequently. coating composition largely preserved the initial composition Along with the degradation, the in vitro bone-bioactivity of designed in the mixture solution. The XRD patterns of the the coatings was assessed by the apatite forming ability in SBF. composite coatings on Ti showed only CH and BGn peaks, and Here, we adopted an acceleration medium, 2Â SBF, to shorten the increasing intensity of glass demonstrated its incorporation the investigation period, which is also widely used to charac- within the coating (Fig. 4b). ATR-FTIR spectra of the terize the in vitro bioactivity of bone repair materials.44,45 Fig. 6 composite coatings also reflected the compositional trend; shows the weight increase of the coatings during the incubation bands related to BGn (544, 1070, 1200, 1365 and 1737 cmÀ1) periods of up to 28 days. For all composite coatings the weight increased as the amount in the coating layer increased. Based on gain was observed as short as 1 day of immersion, while the CH these observations, the CH–BGn composite coatings were coating started to show weight gain in 3 days. The weight gain considered to be easily implemented by the EPD process in was more pronounced as the amount of BGn increased. This terms of possible modulation of coating composition (BGn weight gain was primarily due to the deposition of the apatite content) and thickness, and the coating layers formed were mineral phase onto the coating layer. dense and had uniform thickness. 24948 | J. Mater. Chem., 2012, 22, 24945–24956 This journal is ª The Royal Society of Chemistry 2012
  5. 5. View Article OnlinePublished on 03 October 2012 on http://pubs.rsc.org | doi:10.1039/C2JM33830K Fig. 5 Degradation of the composite coatings in PBS measured for up to 50 days. The weight decrease pattern of the coatings was almost liner with time, suggesting the degradation was mainly associated with surface erosion. Results are mean Æ standard deviation from triplicate samples. The addition of BG nanoparticles (particularly 15% case) accelerated the Downloaded by Dankook University on 23 November 2012 degradation of the coatings. Fig. 6 Weight increase of coatings was observed during incubation of the sample in 2Â SBF for periods of up to 28 days. Results are the mean Æ standard deviation from triplicate samples. The weight gain was ascribed Fig. 4 Characterization of the composite coatings. (a) TG analyses of to the apatite mineral formation on the coatings. The addition of BG the coatings measured up to 900 C, showing weight losses associated nanoparticles significantly enhanced the weight gain, demonstrating with the burning out of organic phases, mainly chitosan. The corre- better apatite forming ability. sponding wt% observed at the plateau after around 500–600 C is meant to be the BG percentage in the composite coatings; 4.89, 9.99 and 14.84% analyzed in 5BGn, 10BGn and 15BGn, respectively. (b) XRD patterns of immersion, and the band intensities also increased with time the coatings on Ti; references of Ti, BGn and CH are also included. (c) FT-IR spectra of the coatings on Ti; reflectance was recorded, and (Fig. 7c). Moreover, the bands at 874 and 1400 cmÀ1 were spectra of CH and BGn are referenced. assigned to v2 C–O and v3 C–O stretching vibration mode of CO32À, signifying the incorporation of a carbonate group in the apatite crystal lattice.48 The results supported the view that the The surface morphology of the samples during the immersion BGn in the composite coatings played significant roles in was observed. 10BGn was presented as the representative sample enhancing the apatite forming ability in SBF, mainly due to the (Fig. 7a). At day 1, some mineral islands were clearly seen on the ionic releases from BGn, which accelerated the supersaturation surface, which covered the whole surface at day 3, and at day 14 of the solution, leading to the precipitation of calcium and the mineralized crystal size became substantially enlarged. A phosphate ions. The CH pure coating also showed an apatite higher magnification of the mineral phase revealed a faceted formation with time, although the apatite forming rate was lower structure of nanocrystallites, as have been typically observed in than the composites. The highly positive-charged amine groups the biomimetically mineralized apatite.46,47 The mineralized in CH attract calcium ions in the medium, accompanied by the phase was further analyzed by XRD (Fig. 7b). The main apatite phosphate ions leading to the mineral formation.49,50 Therefore, peak at 2q ¼ 32 became sharper and more apparent with the accelerated mineralization in the composite coatings may be increasing immersion time. FT-IR spectra also revealed bands ascribed primarily to the enrichment or supersaturation of related to apatite (596, 957, and 1018 cmÀ1 correspond to v2 P–O calcium ions in the medium that are released from the BGn, and bending and v1 P–O and v3 P–O stretching, respectively) after the the consequent ionic precipitation. This journal is ª The Royal Society of Chemistry 2012 J. Mater. Chem., 2012, 22, 24945–24956 | 24949
  6. 6. View Article OnlinePublished on 03 October 2012 on http://pubs.rsc.org | doi:10.1039/C2JM33830K Downloaded by Dankook University on 23 November 2012 Fig. 7 Characterization of the coatings after incubation in 2Â SBF. The 10BGn coating is shown as a representative sample; (a) SEM morphological observation, (b) XRD phase analysis, and (c) FT-IR spectrum change. 2.3. Effects on cell proliferation and osteogenic differentiation up-regulated in the 10BGn coating than the other groups (vs. pure Ti or CH coating), confirming the 10BGn stimulated the The biocompatibility of the EPD composite coatings was osteogenic differentiation process of the MC3T3-E1, particularly addressed by means of in vitro cell responses, including adhesion at 14 days. and proliferation of cells and their osteogenic differentiation. The foregoing results demonstrated that the presence of BGn Pre-osteoblastic MC3T3-E1 cells were cultured on CH or in the coating should primarily be effective in stimulating oste- CH–BGn coatings, and the cell morphology and proliferative ogenic differentiation, rather than the early proliferation. As it is potential were assessed for periods up to 7 days. Fig. 8a shows clear that the coating layer degraded with time (several percent to the SEM morphology of the MC3T3-E1 cells cultured on the 10%) during the culture period of several weeks (Fig. 5), the coatings for 3 days. Coatings of CH and 10BGn are represen- degraded products should affect the cellular responses. Apart tatively shown. Cells adhered and spread well on both coatings, from CH molecules, the ionic products such as calcium and with active cytoplasmic processes. The cell proliferation rate on silicon eluted from the BGn should be the attributes for osteo- the coatings was quantified by means of a CCK assay with genic improvement. The addition of BG particles or eluted ions culture for up to 7 days (Fig. 8b). On-going increase of the CCK from the particles significantly stimulates the osteogenic differ- level with culture time for both coatings was evident for up to 7 entiation, including gene expressions, protein synthesis and days, demonstrating that all the coatings provided favorable mineral formation, either in osteoblastic cells or mesenchymal substrate conditions for the growth of cells without exerting any stem cells.51–53 As the ionic concentrations eluted are of special significant toxic effects. importance in regulating cell behavior, the degraded ionic Having confirmed the cells grew actively on the coatings with products should be in a appropriate range to trigger osteogenic good cell viability, we further sought to examine the effects of development of the cells. In this manner, the composition of the coatings on the osteogenic differentiation of the cells. The BGn should also be modulated to control the ionic elusions; this expression of bone-associated genes, including Col I, ALP, BSP, is not restricted to calcium or silicon, but extends to other trace OPN and OCN, was characterized during culture for 7 and 14 elements that are possibly valuable for the bone regeneration and days, by means of QPCR. The results are depicted in Fig. 9. disease treatment. Herein we observed only gene level (mRNA While the gene expressions were relatively low at day 7, there level by PCR) as an index of osteogenesis, therefore, further were substantial up-regulations at day 14, particularly on the assessments at the protein and calcification/mineralization level 10BGn coating. Except for Col I, which was higher in the CH with prolonged culture periods are considered to be needed to coating, all other genes (ALP, BSP, OPN and OCN) were 24950 | J. Mater. Chem., 2012, 22, 24945–24956 This journal is ª The Royal Society of Chemistry 2012
  7. 7. View Article OnlinePublished on 03 October 2012 on http://pubs.rsc.org | doi:10.1039/C2JM33830K Downloaded by Dankook University on 23 November 2012 Fig. 9 Expression of genes related to bone, including Col I, ALP, BSP, OPN and OCN, was assessed on the cells cultured for periods of 7 and 14 days, by means of QPCR. While the gene expressions were relatively low at day 7, there were substantial up-regulations at day 14, particularly on the 10BGn coating, for all genes (except Col I), resulting in a significance difference with respect to other groups (vs. Ti or CH). weak chemical bonds, and the complexes, under an electric field applied, are considered to be deposited on the metallic substrate, resulting in homogeneous incorporation of ampicillin molecules within the composite coatings. Each coating sample was immersed in PBS at 37 C for different times up to 10–11 weeks to assess the ampicillin release amount using an UV-vis spectrophotometer. Fig. 10 shows the ampicillin release (absolute value) from the coatings of either pure CH (high ampicillin) or 10BGn (low and high ampicillin). The release pattern was smooth (not abrupt) initially, and pre- sented a highly sustained release that was continuous, even up to Fig. 8 (a) SEM morphology of the MC3T3-E1 cells cultured on the 10–11 weeks. Although 10–11 weeks were the maximum time coatings (CH and 10BG, shown as representatives) for 3 days; cells examined herein, the continuing pattern of release at that time adhered and spread well on both coatings, with active cytoplasmic makes it reasonable to suggest that release would continue processes. (b) Cell proliferation assessed by a CCK method for up to 7 beyond this period. This type of release pattern, i.e., long term days demonstrated that all the coatings provided favorable substrate release with almost constant release rate while not showing an conditions for cell growth. Results represented with respect to the Ti initial burst effect, has been considered highly beneficial for use sample (free of coating) at day 1, with mean Æ standard deviation from triplicate samples. of the coatings in biomedical applications, such as coating implants.54–56 Comparing CH and 10BGn coatings, 10BGn exhibited a confirm the full series of osteogenic potential of the BGn in the higher release of ampicillin. Moreover, between the 10BGn coatings. coatings, the sample loaded with higher ampicillin profiled higher release of ampicillin. As to the mechanism of the ampi- cillin release, two phenomena are considered for this kind of 2.4. Drug delivery potential of coatings coating material. One is degradation of the coating layer as was Along with the excellent in vitro bone-bioactivity of the EPD observed in the degradation profile in Fig. 5, with an almost composite coatings, we sought to find out the potential to load linear pattern with time for both coating cases. The other is the and deliver therapeutic molecules. As the model drug, we chose diffusion of ampicillin out through the coating barrier. On closer an antibiotic (Na–ampicillin) and observed the in vitro release examination, the release patterns appeared to show two-stages, profile from the coatings. Ampicillin was added to the EPD consisting of an initial linear step up to $14 days and a parabolic- solution at two different quantities (low; 5 mg or high; 10 mg) in like pattern thereafter. We applied different models for the two- concert with CH or CH–10BGn. After the EPD process, we stages to gain proper fitting of the profiles. One is the zero-order measured the coating weight gain to gauge the quantity of model for the first linear stage up to 14 days; Mt/MN ¼ K0t and material and ampicillin. The negatively charged ampicillin may the other is the Ritger–Peppas empirical equation for the later interact with positively charged BGn or CH molecules to form stage that is to follow the power law;57 Mt/MN ¼ Ktn, where Mt This journal is ª The Royal Society of Chemistry 2012 J. Mater. Chem., 2012, 22, 24945–24956 | 24951
  8. 8. View Article OnlinePublished on 03 October 2012 on http://pubs.rsc.org | doi:10.1039/C2JM33830K Fig. 10 (a) Na–ampicillin was incorporated within the coating layer during the EPD process and the release profile was observed for periods of up to 10–11 weeks. CH and 10BGn coatings were tested representatively. Na–ampicillin was added to the EPD solution in concert with CH or CH–10BG Downloaded by Dankook University on 23 November 2012 nanoparticles; at two different concentrations (low 5 mg and high 10 mg; CH ¼ 100 mg and BG ¼ 10 mg). After the EPD process (40 kV, 5 min), the coating layer was gently washed and dried and the sample was immersed in PBS at 37 C for different time points, prior to an assay for the ampicillin release amount using a UV-vis spectrophotometer. A continuous and highly sustained release for up to the period tested (10–11 weeks) was recorded. Data well fitted according to the combined model of the zero-order model (initial stage) and Ritger–Peppas empirical equation (later stage), and parameters are summarized in Table 1. (b) Antibacterial tests of the ampicillin-loaded 10BGn coating against Streptococcus mutants using an agar diffusion plate. Antibacterial effective zone was formed around the ampicillin-loaded coating at 24 h and was maintained and even increased for up to 5 days (time point for the bacteria lifespan), which was however not observed in the coating without ampicillin loading, confirming the efficacy of the drug delivery through the composite coating layer. Representative images of the agar diffusion test are shown for comparison purpose (1 and 5 days of ampicillin-added 10BGn vs. 1 day of ampicillin-free 10BGn). and MN are the absolute amount of drug released at time t and as hydrogels, swelling polymers and semi-interpenetrating infinite time (N), respectively, and K0 and K are released rate networks.62–64 constants for each equation, incorporating structural and The initial drug release may be mainly ascribed to the degra- geometric characteristics of the drug delivery device, and n is the dation (surface erosion) of the coating layer as the surface- released exponent, indicative of the drug release mechanism. The reaction (erosion) process has a linear dependence on time; parameters determined from the curve fittings are summarized in although a level of diffusion out of drug also occurred, the Table 1. Although the models are simplified forms without degradation will be the rate determinant. The slightly faster considering the moving boundary problems as our coatings are release of the drug in the 10BGn was also associated with the degradable and thus do not preserve constant volumes, they more rapid coating degradation in the sample. However, after a should allow the interpretation of the drug release kinetics in a certain period ($14 days), when a depletion zone of drug was much easier and simpler way, as have generally been carried out created at the surface region, drugs below the zone could be elsewhere.58–61 The initial stage was shown to follow well the released mainly by a diffusion through the surface coating layer, linearity, with the R2 value lower than 0.99. The 2nd stage also which would be evident as the curved parabolic-like pattern at a showed a power exponent of 0.44, 0.37 and 0.38, for CH (high), longer period. Although the surface erosion is processed, and at 10BGn (low) and 10BGn (high) coating, respectively, values this step the drugs existing at the eroding surface should be lower than 0.5 (an index of the diffusion-controlled process), released, drugs in the deeper region could still be diffusing out suggesting the stage is a sort of anomalous diffusion-controlled through the barrier of the coating layer. As the drug release (slight deviation from Fickian diffusion-controlled) release process results from a complex of the coating degradation and phenomenon, which has been reported elsewhere, systems such the diffusion through coating layer, the outcome pattern with respect to time will not be an abrupt transition, but rather might be a smooth pattern. Coating degradability, interactions between Table 1 Summary of release-model parameters (K0, K, and n), defining drug molecules and coating materials, and permeability or the release mechanism of the drug from the coatings. Linear release with diffusivity of drugs through the coating can significantly influ- zero-order kinetics; Mt/MN ¼ K0t at the 1st stage, and then at the 2nd stage with a power law relationship provided empirically by Ritger– ence the drug release profile. These aspects need to be considered Peppas; Mt/MN ¼ Ktn carefully in the design of coatings to control the drug release profile. Although this release pattern may not be applicable in Coating sample with ampicillin parallel to all other types of drugs, because of the difference in Model Parameter CH (low) 10BGn (low) 10BGn (high) the drug size and interactions with coating materials, particularly for small hydrophilic (or possibly anionic) drugs this long-term Zero-order model K0 2.82 3.38 4.16 (over 2–3 months) sustained release can be envisioned. As the Ritger–Peppas K 17.5 22.6 35.2 empirical model n 0.44 0.37 0.38 ampicillin molecules are anionic-charged, a sort of weak charge– charge interactions is possible with the BGn or CH molecules 24952 | J. Mater. Chem., 2012, 22, 24945–24956 This journal is ª The Royal Society of Chemistry 2012
  9. 9. View Article Online within the coating layer, which might favor a slow diffusion basis), tetraethyl orthosilicate (TEOS, C8H20O4Si, 98%), meth- release. anol anhydrous (CH4O, 99.8%), toluene anhydrous (C7H8, Having confirmed that ampicillin incorporated within the 99.8%), and 3-aminopropyl triethoxysilane (APTES, coating layer was released in a fairly sustained manner, we next C9H23NO3Si, $98%) were purchased from Sigma-Aldrich designed an experiment to observe the antibacterial effects (USA) and were used as-received without any further against Streptococcus mutants, as this is one of the major and purification. well-recognized oral bacteria and thus has been carefully researched in dental implantations. As the ampicillin release 4.2. Synthesis of BG nanoparticles and surface patterns of the coatings were similar, we chose 10BGn as a functionalization representative sample group. We placed the bacteria on the agar diffusion plate and then introduced the 10BGn coating sample BG nanoparticles for the EPD coating were prepared by a sol–gel with or without ampicillin. The antibacterial effective zone technique. The Si/Ca ratio of the sol–gel solution was set at 85/15 formed around the sample was examined every 24 h for up to 5 in mol% to achieve a binary composition of sol–gel glass 85SiO2–Published on 03 October 2012 on http://pubs.rsc.org | doi:10.1039/C2JM33830K days (time point generally accepted for the bacteria lifespan). 15CaO. From a pilot study, the 15CaO has shown excellent Clearly, the effective zone was formed around the ampicillin- in vitro bioactivity while preserving better spherical nanoparticle loaded coating sample at 24 h, which was well-maintained and morphology than other compositions (5CaO and 25CaO). For even slightly enhanced up to 5 days. However, there was no zone this, 5 g PEG was dissolved in 150 ml of ethanol while vigorous stirring at 40 C, and then 30 ml of ammonium Downloaded by Dankook University on 23 November 2012 formation in the drug-free coating sample. The results demon- strated the effective role of the ampicillin released from the hydroxide and 358 g of Ca(NO3)2$4H2O was added until a coating layer. transparent mixture was obtained. Another solution of 2 ml Further work is needed to assess the long-term (weeks to TEOS in 20 ml ethanol was prepared, which was added to the months) delivery potential of the currently developed coating above solution dropwise and then homogenized using a sonor- system such as delivery of growth factors, and the consequent eactor (LH700S ultra-sonic generator; Ulsso Hitech, Korea) at effects on cell proliferation and osteogenic differentiation. In 20 kHz and 700 W ultrasonication (35% power for 10 min, with tandem with the process advantages such as simplicity and ease an on/off cycle of 10 s/10 s). The output power was 220 W in a 10 set-up, and accessibility to complex-shaped metal scaffolds, the s on/10 s off cycle for 20 min. A vigorous stirring of the mixture currently engineered CH–BGn composite EPD coatings proved solution for 24 h at room temperature produced a white gel compositional merits like excellent bone-bioactivity and osteo- precipitate, which was then centrifuged at 10 000 rpm and genic stimulatory effects, and capacity to long-term deliver washed with distilled water and ethanol, and filtered. The white therapeutic molecules. These facts indicate the potential useful- powder was heat-treated at 600 C for 5 h to obtain BG ness of the coatings on implants or scaffolds for bone repair and nanoparticles. regeneration. The surface of BG nanoparticles was functionalized with amine groups using APTES. BG nanoparticles of 0.1 g were 3. Conclusions added to 50 ml toluene and sonicated for 30 min to a homoge- neous solution. One milliliter of APTES was added to this Composites of CH and BGn up to 20 wt% were electrophoreti- solution and then refluxed at 80 C for 24 h, which was followed cally deposited onto Ti uniformly with thicknesses of $10–50 by a centrifugation at 10 000 rpm for 5 min and stringent mm. The incorporation of BGn increased the coating weight gain washing with toluene and ethanol. The product was dried in an and the degradation was also increased in the composite coat- oven at 80 C for 24 h. ings. The BGn present in the coatings significantly improved the The morphology of the BG nanoparticles was observed by in vitro apatite forming ability and osteogenic differentiation of transmission electron microscopy (TEM). The chemical bond cells. Furthermore, a therapeutic drug (ampicillin used as model structure of the nanoparticles was analyzed by Fourier transform drug) effectively incorporated during the coating process was infrared (FT-IR; Varian 640-IR). The phase was characterized shown to have a sustained release for over 10–11 weeks. The with X-ray diffraction (XRD; Ultima IV, Rigaku). The surface effects of the drug release were confirmed by an antibacterial test electrical potential of the nanoparticles was analyzed by a zeta against Streptococcus mutants. Along with the processing aspects (z)-potential measurement (Zetasizer Nano, Malvern, UK) at of the EPD, the compositional merits of the CH–BGn allow a 25 C. The instrument determines the electrophoretic mobility of range of potential applications for coatings of metallic implants the particles automatically and converts it to the z-potential and scaffolds for bone repair and regeneration. using a Smoluchowski’s equation. 4. Experimental conditions 4.3. Suspensions and EPD process 4.1. Materials For the success of the EPD process, it is essential to prepare a Commercial pure titanium (Ti) (cp Ti, Senulbio Biotech, Korea) stable suspension. CH dissolved in a 1% acetic acid solution was in a rectangular plate form (10 mm  10 mm  1 mm) was used dispersed at 1 g lÀ1 in an ethanol–water co-solvent (25% v/v for the coatings. Medium molecular weight CH (Mw ¼ 200 000 water). Within the CH solution, aminated BG nanoparticles were Da, deacetylation degree of about 85%), acetic acid ($99%), dispersed by ultrasonification for 30 min at varying concentra- poly(ethylene glycol) (PEG, (C2H4)nH2O, Mn: 10 000), tions; 5, 10, 15 and 20 wt%. The homogeneous dispersion of the Ca(NO3)2$4H2O, NH4OH (28% NH3 in water, $99.99% metal BG nanoparticles within CH solution was confirmed by means of This journal is ª The Royal Society of Chemistry 2012 J. Mater. Chem., 2012, 22, 24945–24956 | 24953

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