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Chitosan–nanobioactive glass electrophoretic coatings with bone regenerativeand 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*abc

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

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

Commercial pure titanium (CPTi) and its alloys have been

extensively used as implants in dental, cranial-maxillary facial

reconstruction and orthopedic applications.1 This is primarily

due to their excellent corrosion resistance and biocompatibility,

allowing bone-implant integration.2,3 The biocompatibility of

metallic implants can be improved by the surface modification,

such as the control over roughness and topography, and the

coating with bioactive compositions. While the coatings are the

protective layer against corrosion of metals, they impart new

compositions to the surface, allowing a large spectrum of

possibilities of choosing compositions to trigger proper tissue

reactions. A number of coating techniques have been developed,

which include plasma spraying, anodic oxidation, sol–gel

aInstitute of Tissue Regeneration Engineering (ITREN), DankookUniversity, South Korea. E-mail: kimhw@dku.edu; Fax: +82 41 5503085; Tel: +82 41 550 3081bDepartment of Nanobiomedical Science & WCU Research Center,Dankook University Graduate School, South KoreacDepartment of Biomaterials Science, College of Dentistry, DankookUniversity, South Korea

This journal is ª The Royal Society of Chemistry 2012

process, biomimetic coating, sputtering and electrochemical

treatment.4–11

Electrophoretic deposition (EPD) is one of the most useful and

effective coating methods available, mainly due to its simplicity

and low cost. Advantages also include the possibility of

producing a coating layer with high uniformity and variable

thickness (0.3–100 mm), the capacity to coat complex shapes, the

ease of control over the coating composition and commercial

availability. It is possible to apply either an anodic or cathodic

treatment depending on the charge of the particles or molecules

being deposited.9 Using the EPD method, a range of composi-

tions, including biopolymers,9,12,13 bioactive ceramics14,15 and

composites16–21 have been deposited for biomedical implants.

Among the compositions, here we focus on biopolymer

composites with bioactive inorganic nanoparticles. In fact, there

has been significant attention to produce biopolymer composite

coatings with inorganic particles by the EPD method.17–23 Inor-

ganic particles, including hydroxyapatite (HA), carbon nano-

tube, silica, and their combinations, introduced into the

polymeric solutions, were enabled to form co-deposits by the

EPD process. Among the biopolymer sources, chitosan (CH) has

been widely used, as it is biocompatible and degradable and is

J. Mater. Chem., 2012, 22, 24945–24956 | 24945

Fig. 1 Characteristics of BGn; (a) XRD pattern, (b) FT-IR spectra

before and after amination, (c) z-potentials before and after amination,

and (d) TEM image, and the colloidal solution of BGn in CH; (e) TEM

image after drying and (f) turbidity test monitored over 24 h.

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highly positively charged, allowing for the ease of cathodic EPD.

For the bioactive inorganic component, here we used novel

inorganic nanoparticles, bioactive glass nanoparticles (BGn),

which were newly developed in this study. BGn are considered to

disperse well in the CH-containing acidic solution and conse-

quently provide excellent bone-bioactivity to the coating layer,

thus presenting the potential for bone regeneration.

CH is a natural polymer that can be obtained from the

exoskeleton of insects, crustaceans and fungi.22 It is generally

obtained by deacetylation of its parent polymer chitin, a poly-

saccharide that is widely distributed in nature. While the parent

chitin is insoluble in most organic solvents, CH is readily soluble

in dilute acidic solutions below pH �6.0 due to the quaternisa-

tion of the amine groups that have a pKa value of 6.3, which

allows CH to be a water-soluble cationic polyelectrolyte.24

Because of the biocompatibility and charged property, CH has

been used as biomedical materials, including scaffolds, gene

delivery systems and coating materials.21–28 Particularly for EPD

coating, CH molecules are considered effective for deposition

under cathodic EPD conditions due to its positively charged

nature.

The BGn used in this study were sourced from a sol–gel

precursor and prepared using a surfactant-mediated emulsifica-

tion method. In fact, the class of BGs has long been considered

one of the most potential bioactive inorganic materials in bone

regeneration areas since the advent of melt-derived composi-

tions.29–33 More recently, the nano-sized (generally tens to

hundreds of nanometers) forms of BGs such as nanofibers and

nanoparticles have been developed in anticipation of further

potential applications, including nanocomposites with poly-

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

carried out to ascertain the efficacy, to provide insight into the

use of other bioactive molecules more relevant to bone repair

and regeneration.

24946 | J. Mater. Chem., 2012, 22, 24945–24956

2. Results and discussion

2.1. Properties of CH–BGn coatings

Fig. 1 shows the characteristics of the BGn prepared for the

coating materials for EPD. A typical amorphous silica phase

with only one broad peak at 2q ¼ 22.5� was noticed in the XRD

pattern (Fig. 1a). The BGn were functionalized with amine

groups using APTES to allow cathodic EPD coating. While the

FTIR spectrum of non-functionalized BGn displays bands

related to the silica glass such as 544 and 1200 cm�1 (Si–O–Si

bending), 1070 cm�1 (Si–O–Si stretching) and 784 cm�1 (Si–O–

Ca vibration),40,41 additional bands at 1365 and 1737 cm�1

assigned to –NH2 stretching mode of aromatic amine also

appeared after the amine-functionalization42 (Fig. 1b). The

z-potential of the BGn measured at pH 7.4 changed from highly

negative (�24.9 mV) to positive (+21.9 mV) after the amination,

confirming the successful amine-functionalization of the surface

(Fig. 1c). Furthermore, the z-potentials of amine-functionalized

BGn measured at pH 3–4 (the pH range of EPD solutions)

showed much higher positive values (from +24 mV to +31 mV).

The TEM morphology of BGn showed the development of

uniform-sized particles less than a hundred nanometers (85 �15 nm, Fig. 1d). Prior to the EPD process, we observed the

properties of the BGn–CH solution. A drop of the solution

(10BGn–CH) was dried on a copper grid and the TEM image

was taken (Fig. 1e). Nanoparticles easily came close to each other

during the drying process, and the individual nanoparticles were

separated completely enclosed by the CH matrix. The colloidal

stability of the solution was also assessed by a turbidity test. An

optical transmission % of the solution was monitored every 1 h

This journal is ª The Royal Society of Chemistry 2012

Fig. 2 Weight gains of samples during EPD coating measured at varying

coating parameters, including pH, voltage and time: (a) for 10BGn at two

different pHs (3.1 and 3.6) as a function of voltage, (b) for CH and

10BGn at 60 V and pH 3.6, as a function of time, and (c) for all

compositions at 5 min with varying voltage.

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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.

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

colloidal particles moved towards the cathode to be neutralized

by consuming the hydroxyl groups (OH�) 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 becamemore 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

linear as a function of time (Fig. 2b). An observation of the

weight gain at different compositions (at 5 min coating time)

revealed that the incorporation of BGn increased the coating

weight (Fig. 2c). The weight increase as a function of the amount

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

thickness) may explain this. All the coatings produced herein

stably adhered to the metallic substrate, not allowing the ease of

scratching and peeling off, and delamination even after the

ultrasonic vibration in water. More in-depth tests on mechanical

properties of the coatings will be discussed in future works.

At this point a possible EPD mechanism of the BGn–CH

composite is proposed. In acidic solution, CH molecules become

positively charged by protonation, and thus easily accumulate at

the electrode by the electrophoresis.22 Moreover, the BGn, as

they are also positively charged, can also deposit similarly,

resulting in co-deposition with CH. In fact, the cathodic depo-

sition of CH composites either with silica or hydroxyapatite

(HA) has been reported elsewhere.20,21,23 In those cases, the silica

or HA particles are initially negatively charged, which however

become positively charged due to the adsorption of CH mole-

cules and thus co-deposit with CH.

The coating morphology was observed by SEM, as shown in

Fig. 3. While the pure CH coating showed homogenous and

clean morphology (Fig. 3a), the composite coatings had a rough

This journal is ª The Royal Society of Chemistry 2012

morphology and this was more pronounced as the amount of

BGn increased (Fig. 3b–d). The BGn appeared to be clustered,

contrasted in brighter areas with localized sizes of around a few

micrometers (larger than individual BGn). This cluster-like

formation of BGn is considered to result from the EPD process

as the BGn present in the CH solution are relatively stable. The

electric field applied should alter the surface electrostatic status

of the BGn, possibly weakening the stability of individual

nanoparticles and rendering them to form cluster-like areas in

the coating layer with sizes of a few micrometers. The literature

also reported a similar phenomenon for the clusters of nano-

particles.23,43 Strictly speaking, the BGn in the clusters should,

however, be separated, surrounded by the CH molecules,

moreover the clustered areas appeared to distribute at similar

spatial distances throughout the coating layer – a feature not

readily found in the composite coatings where micron-sized

particles were initially used. However, strategies to improve the

nanoparticle dispersion in the EPD coating layer will be required

for further studies, and the possible ways are to provide a

stronger positive charge to the nanoparticles or to decrease the

J. Mater. Chem., 2012, 22, 24945–24956 | 24947

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.

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content of nanoparticles. The cross-section morphology of the

coating layer was examined by scratching off from the Ti

substrate (Fig. 3e); a thickness of �15 mm was formed in the

5BGn coating. Similarly observed thickness was �12 mm for CH

coating, �30 mm for 10BGn coating and �48 mm for the 15BGn

coating, which corresponded well to the results of the coating

weight gain shown in Fig. 2c.

The EPD coatings were further characterized, as presented in

Fig. 4. The exact composition of the composite coatings was

investigated by TG analysis. For this, some parts of the coating

layer scratched off from each sample were heat-treated up to

900 �C and the weight change was monitored (Fig. 4a). The

TGA pattern of CH showed three steps in weight loss; first 22%

up to �200 �C was attributed to the liberation of adsorbed

water, and two further steps at 200–350 �C and 350–600 �Cwere from the thermal degradation of CH. Whilst CH showed

almost 100% weight loss at �600 �C, all the composite coatings

preserved a certain amount of weight at the end, although the

three-step behavior was similarly observed. The remaining

weight measured was 4.89, 9.99 and 14.84% for 5BGn, 10BGn

and 15BGn coating, respectively. The results confirmed that the

coating composition largely preserved the initial composition

designed in the mixture solution. The XRD patterns of the

composite coatings on Ti showed only CH and BGn peaks, and

the increasing intensity of glass demonstrated its incorporation

within the coating (Fig. 4b). ATR-FTIR spectra of the

composite coatings also reflected the compositional trend;

bands related to BGn (544, 1070, 1200, 1365 and 1737 cm�1)

increased as the amount in the coating layer increased. Based on

these observations, the CH–BGn composite coatings were

considered to be easily implemented by the EPD process in

terms of possible modulation of coating composition (BGn

content) and thickness, and the coating layers formed were

dense and had uniform thickness.

24948 | J. Mater. Chem., 2012, 22, 24945–24956

2.2. In vitro degradation and apatite forming ability

Some important in vitro properties of the coating layers for the

hard tissue applications, including degradation and bone-

bioactivity, were also investigated. Fig. 5 shows the degradation

of the coatings with time during incubation in PBS at 37 �C for

periods of up to 50 days. For all coating compositions, the

degradation profile was almost liner with time, and the incor-

poration of BGn increased the degradation rate. For the CH

coating, the degradation was �5% for 7 days, �13% for 21 days,

�18% for 35 days and 34% for 50 days. For the 15BGn coating,

the degradation was �12% for 7 days, �25% for 21 days, �32%

for 35 days and �42% for 35 days. The incorporation of BGn is

thus considered to speed up the hydrolytic degradation of the

coatings, in the form of ionic release of the BGn and/or disso-

ciation of CH molecules. One interesting thing was the linear

release pattern observed in the coatings, which is consistent with

the view that the coating degradation is primarily associated with

the surface erosion process. It is envisaged that the degradation

process should significantly influence the release pattern of drugs

that are incorporated within the coating layer, as discussed

subsequently.

Along with the degradation, the in vitro bone-bioactivity of

the coatings was assessed by the apatite forming ability in SBF.

Here, we adopted an acceleration medium, 2� SBF, to shorten

the investigation period, which is also widely used to charac-

terize the in vitro bioactivity of bone repair materials.44,45 Fig. 6

shows the weight increase of the coatings during the incubation

periods of up to 28 days. For all composite coatings the weight

gain was observed as short as 1 day of immersion, while the CH

coating started to show weight gain in 3 days. The weight gain

was more pronounced as the amount of BGn increased. This

weight gain was primarily due to the deposition of the apatite

mineral phase onto the coating layer.

This journal is ª The Royal Society of Chemistry 2012

Fig. 4 Characterization of the composite coatings. (a) TG analyses of

the coatings measured up to 900 �C, showing weight losses associated

with the burning out of organic phases, mainly chitosan. The corre-

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

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

spectra of CH and BGn are referenced.

Fig. 5 Degradation of the composite coatings in PBSmeasured 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 � standard deviation from triplicate samples.

The addition of BG nanoparticles (particularly 15% case) accelerated the

degradation of the coatings.

Fig. 6 Weight increase of coatings was observed during incubation of

the sample in 2� SBF for periods of up to 28 days. Results are the mean�standard deviation from triplicate samples. The weight gain was ascribed

to the apatite mineral formation on the coatings. The addition of BG

nanoparticles significantly enhanced the weight gain, demonstrating

better apatite forming ability.

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The surface morphology of the samples during the immersion

was observed. 10BGn was presented as the representative sample

(Fig. 7a). At day 1, some mineral islands were clearly seen on the

surface, which covered the whole surface at day 3, and at day 14

the mineralized crystal size became substantially enlarged. A

higher magnification of the mineral phase revealed a faceted

structure of nanocrystallites, as have been typically observed in

the biomimetically mineralized apatite.46,47 The mineralized

phase was further analyzed by XRD (Fig. 7b). The main apatite

peak at 2q ¼ 32� became sharper and more apparent with

increasing immersion time. FT-IR spectra also revealed bands

related to apatite (596, 957, and 1018 cm�1 correspond to v2 P–O

bending and v1 P–O and v3 P–O stretching, respectively) after the

This journal is ª The Royal Society of Chemistry 2012

immersion, and the band intensities also increased with time

(Fig. 7c). Moreover, the bands at 874 and 1400 cm�1 were

assigned to v2 C–O and v3 C–O stretching vibration mode of

CO32�, signifying the incorporation of a carbonate group in the

apatite crystal lattice.48 The results supported the view that the

BGn in the composite coatings played significant roles in

enhancing the apatite forming ability in SBF, mainly due to the

ionic releases from BGn, which accelerated the supersaturation

of the solution, leading to the precipitation of calcium and

phosphate ions. The CH pure coating also showed an apatite

formation with time, although the apatite forming rate was lower

than the composites. The highly positive-charged amine groups

in CH attract calcium ions in the medium, accompanied by the

phosphate ions leading to the mineral formation.49,50 Therefore,

the accelerated mineralization in the composite coatings may be

ascribed primarily to the enrichment or supersaturation of

calcium ions in the medium that are released from the BGn, and

the consequent ionic precipitation.

J. Mater. Chem., 2012, 22, 24945–24956 | 24949

Fig. 7 Characterization of the coatings after incubation in 2� SBF. The 10BGn coating is shown as a representative sample; (a) SEM morphological

observation, (b) XRD phase analysis, and (c) FT-IR spectrum change.

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2.3. Effects on cell proliferation and osteogenic differentiation

The biocompatibility of the EPD composite coatings was

addressed by means of in vitro cell responses, including adhesion

and proliferation of cells and their osteogenic differentiation.

Pre-osteoblastic MC3T3-E1 cells were cultured on CH or

CH–BGn coatings, and the cell morphology and proliferative

potential were assessed for periods up to 7 days. Fig. 8a shows

the SEM morphology of the MC3T3-E1 cells cultured on the

coatings for 3 days. Coatings of CH and 10BGn are represen-

tatively shown. Cells adhered and spread well on both coatings,

with active cytoplasmic processes. The cell proliferation rate on

the coatings was quantified by means of a CCK assay with

culture for up to 7 days (Fig. 8b). On-going increase of the CCK

level with culture time for both coatings was evident for up to 7

days, demonstrating that all the coatings provided favorable

substrate conditions for the growth of cells without exerting any

significant toxic effects.

Having confirmed the cells grew actively on the coatings with

good cell viability, we further sought to examine the effects of

coatings on the osteogenic differentiation of the cells. The

expression of bone-associated genes, including Col I, ALP, BSP,

OPN and OCN, was characterized during culture for 7 and 14

days, by means of QPCR. The results are depicted in Fig. 9.

While the gene expressions were relatively low at day 7, there

were substantial up-regulations at day 14, particularly on the

10BGn coating. Except for Col I, which was higher in the CH

coating, all other genes (ALP, BSP, OPN and OCN) were

24950 | J. Mater. Chem., 2012, 22, 24945–24956

up-regulated in the 10BGn coating than the other groups (vs.

pure Ti or CH coating), confirming the 10BGn stimulated the

osteogenic differentiation process of the MC3T3-E1, particularly

at 14 days.

The foregoing results demonstrated that the presence of BGn

in the coating should primarily be effective in stimulating oste-

ogenic differentiation, rather than the early proliferation. As it is

clear that the coating layer degraded with time (several percent to

10%) during the culture period of several weeks (Fig. 5), the

degraded products should affect the cellular responses. Apart

from CH molecules, the ionic products such as calcium and

silicon eluted from the BGn should be the attributes for osteo-

genic improvement. The addition of BG particles or eluted ions

from the particles significantly stimulates the osteogenic differ-

entiation, including gene expressions, protein synthesis and

mineral formation, either in osteoblastic cells or mesenchymal

stem cells.51–53 As the ionic concentrations eluted are of special

importance in regulating cell behavior, the degraded ionic

products should be in a appropriate range to trigger osteogenic

development of the cells. In this manner, the composition of the

BGn should also be modulated to control the ionic elusions; this

is not restricted to calcium or silicon, but extends to other trace

elements that are possibly valuable for the bone regeneration and

disease treatment. Herein we observed only gene level (mRNA

level by PCR) as an index of osteogenesis, therefore, further

assessments at the protein and calcification/mineralization level

with prolonged culture periods are considered to be needed to

This journal is ª The Royal Society of Chemistry 2012

Fig. 8 (a) SEM morphology of the MC3T3-E1 cells cultured on the

coatings (CH and 10BG, shown as representatives) for 3 days; cells

adhered and spread well on both coatings, with active cytoplasmic

processes. (b) Cell proliferation assessed by a CCK method for up to 7

days demonstrated that all the coatings provided favorable substrate

conditions for cell growth. Results represented with respect to the Ti

sample (free of coating) at day 1, with mean � standard deviation from

triplicate samples.

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).

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confirm the full series of osteogenic potential of the BGn in the

coatings.

2.4. Drug delivery potential of coatings

Along with the excellent in vitro bone-bioactivity of the EPD

composite coatings, we sought to find out the potential to load

and deliver therapeutic molecules. As the model drug, we chose

an antibiotic (Na–ampicillin) and observed the in vitro release

profile from the coatings. Ampicillin was added to the EPD

solution at two different quantities (low; 5 mg or high; 10 mg) in

concert with CH or CH–10BGn. After the EPD process, we

measured the coating weight gain to gauge the quantity of

material and ampicillin. The negatively charged ampicillin may

interact with positively charged BGn or CH molecules to form

This journal is ª The Royal Society of Chemistry 2012

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

10–11 weeks. Although 10–11 weeks were the maximum time

examined herein, the continuing pattern of release at that time

makes it reasonable to suggest that release would continue

beyond this period. This type of release pattern, i.e., long term

release with almost constant release rate while not showing an

initial burst effect, has been considered highly beneficial for use

of the coatings in biomedical applications, such as coating

implants.54–56

Comparing CH and 10BGn coatings, 10BGn exhibited a

higher release of ampicillin. Moreover, between the 10BGn

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

coating material. One is degradation of the coating layer as was

observed in the degradation profile in Fig. 5, with an almost

linear pattern with time for both coating cases. The other is the

diffusion of ampicillin out through the coating barrier. On closer

examination, the release patterns appeared to show two-stages,

consisting of an initial linear step up to�14 days and a parabolic-

like pattern thereafter. We applied different models for the two-

stages to gain proper fitting of the profiles. One is the zero-order

model for the first linear stage up to 14 days; Mt/MN ¼ K0t and

the other is the Ritger–Peppas empirical equation for the later

stage that is to follow the power law;57 Mt/MN ¼ Ktn, where Mt

J. Mater. Chem., 2012, 22, 24945–24956 | 24951

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

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).

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and MN are the absolute amount of drug released at time t and

infinite time (N), respectively, and K0 and K are released rate

constants for each equation, incorporating structural and

geometric characteristics of the drug delivery device, and n is the

released exponent, indicative of the drug release mechanism. The

parameters determined from the curve fittings are summarized in

Table 1. Although the models are simplified forms without

considering the moving boundary problems as our coatings are

degradable and thus do not preserve constant volumes, they

should allow the interpretation of the drug release kinetics in a

much easier and simpler way, as have generally been carried out

elsewhere.58–61 The initial stage was shown to follow well the

linearity, with the R2 value lower than 0.99. The 2nd stage also

showed a power exponent of 0.44, 0.37 and 0.38, for CH (high),

10BGn (low) and 10BGn (high) coating, respectively, values

lower than 0.5 (an index of the diffusion-controlled process),

suggesting the stage is a sort of anomalous diffusion-controlled

(slight deviation from Fickian diffusion-controlled) release

phenomenon, which has been reported elsewhere, systems such

Table 1 Summary of release-model parameters (K0, K, and n), definingthe release mechanism of the drug from the coatings. Linear release withzero-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–Peppas; Mt/MN ¼ Ktn

Model Parameter

Coating sample with ampicillin

CH (low) 10BGn (low) 10BGn (high)

Zero-order model K0 2.82 3.38 4.16Ritger–Peppasempirical model

K 17.5 22.6 35.2n 0.44 0.37 0.38

24952 | J. Mater. Chem., 2012, 22, 24945–24956

as hydrogels, swelling polymers and semi-interpenetrating

networks.62–64

The initial drug release may be mainly ascribed to the degra-

dation (surface erosion) of the coating layer as the surface-

reaction (erosion) process has a linear dependence on time;

although a level of diffusion out of drug also occurred, the

degradation will be the rate determinant. The slightly faster

release of the drug in the 10BGn was also associated with the

more rapid coating degradation in the sample. However, after a

certain period (�14 days), when a depletion zone of drug was

created at the surface region, drugs below the zone could be

released mainly by a diffusion through the surface coating layer,

which would be evident as the curved parabolic-like pattern at a

longer period. Although the surface erosion is processed, and at

this step the drugs existing at the eroding surface should be

released, drugs in the deeper region could still be diffusing out

through the barrier of the coating layer. As the drug release

process results from a complex of the coating degradation and

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

drug molecules and coating materials, and permeability or

diffusivity of drugs through the coating can significantly influ-

ence the drug release profile. These aspects need to be considered

carefully in the design of coatings to control the drug release

profile. Although this release pattern may not be applicable in

parallel to all other types of drugs, because of the difference in

the drug size and interactions with coating materials, particularly

for small hydrophilic (or possibly anionic) drugs this long-term

(over 2–3 months) sustained release can be envisioned. As the

ampicillin molecules are anionic-charged, a sort of weak charge–

charge interactions is possible with the BGn or CH molecules

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within the coating layer, which might favor a slow diffusion

release.

Having confirmed that ampicillin incorporated within the

coating layer was released in a fairly sustained manner, we next

designed an experiment to observe the antibacterial effects

against Streptococcus mutants, as this is one of the major and

well-recognized oral bacteria and thus has been carefully

researched in dental implantations. As the ampicillin release

patterns of the coatings were similar, we chose 10BGn as a

representative sample group. We placed the bacteria on the agar

diffusion plate and then introduced the 10BGn coating sample

with or without ampicillin. The antibacterial effective zone

formed around the sample was examined every 24 h for up to 5

days (time point generally accepted for the bacteria lifespan).

Clearly, the effective zone was formed around the ampicillin-

loaded coating sample at 24 h, which was well-maintained and

even slightly enhanced up to 5 days. However, there was no zone

formation in the drug-free coating sample. The results demon-

strated the effective role of the ampicillin released from the

coating layer.

Further work is needed to assess the long-term (weeks to

months) delivery potential of the currently developed coating

system such as delivery of growth factors, and the consequent

effects on cell proliferation and osteogenic differentiation. In

tandem with the process advantages such as simplicity and ease

set-up, and accessibility to complex-shaped metal scaffolds, the

currently engineered CH–BGn composite EPD coatings proved

compositional merits like excellent bone-bioactivity and osteo-

genic stimulatory effects, and capacity to long-term deliver

therapeutic molecules. These facts indicate the potential useful-

ness of the coatings on implants or scaffolds for bone repair and

regeneration.

3. Conclusions

Composites of CH and BGn up to 20 wt% were electrophoreti-

cally deposited onto Ti uniformly with thicknesses of �10–50

mm. The incorporation of BGn increased the coating weight gain

and the degradation was also increased in the composite coat-

ings. The BGn present in the coatings significantly improved the

in vitro apatite forming ability and osteogenic differentiation of

cells. Furthermore, a therapeutic drug (ampicillin used as model

drug) effectively incorporated during the coating process was

shown to have a sustained release for over 10–11 weeks. The

effects of the drug release were confirmed by an antibacterial test

against Streptococcus mutants. Along with the processing aspects

of the EPD, the compositional merits of the CH–BGn allow a

range of potential applications for coatings of metallic implants

and scaffolds for bone repair and regeneration.

4. Experimental conditions

4.1. Materials

Commercial pure titanium (Ti) (cp Ti, Senulbio Biotech, Korea)

in a rectangular plate form (10 mm � 10 mm � 1 mm) was used

for the coatings. Medium molecular weight CH (Mw ¼ 200 000

Da, deacetylation degree of about 85%), acetic acid ($99%),

poly(ethylene glycol) (PEG, (C2H4)nH2O, Mn: 10 000),

Ca(NO3)2$4H2O, NH4OH (28% NH3 in water, $99.99% metal

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basis), tetraethyl orthosilicate (TEOS, C8H20O4Si, 98%), meth-

anol anhydrous (CH4O, 99.8%), toluene anhydrous (C7H8,

99.8%), and 3-aminopropyl triethoxysilane (APTES,

C9H23NO3Si, $98%) were purchased from Sigma-Aldrich

(USA) and were used as-received without any further

purification.

4.2. Synthesis of BG nanoparticles and surface

functionalization

BG nanoparticles for the EPD coating were prepared by a sol–gel

technique. The Si/Ca ratio of the sol–gel solution was set at 85/15

in mol% to achieve a binary composition of sol–gel glass 85SiO2–

15CaO. From a pilot study, the 15CaO has shown excellent

in vitro bioactivity while preserving better spherical nanoparticle

morphology than other compositions (5CaO and 25CaO). For

this, 5 g PEG was dissolved in 150 ml of ethanol while

vigorous stirring at 40 �C, and then 30 ml of ammonium

hydroxide and 358 g of Ca(NO3)2$4H2O was added until a

transparent mixture was obtained. Another solution of 2 ml

TEOS in 20 ml ethanol was prepared, which was added to the

above solution dropwise and then homogenized using a sonor-

eactor (LH700S ultra-sonic generator; Ulsso Hitech, Korea) at

20 kHz and 700 W ultrasonication (35% power for 10 min, with

an on/off cycle of 10 s/10 s). The output power was 220 W in a 10

s on/10 s off cycle for 20 min. A vigorous stirring of the mixture

solution for 24 h at room temperature produced a white gel

precipitate, which was then centrifuged at 10 000 rpm and

washed with distilled water and ethanol, and filtered. The white

powder was heat-treated at 600 �C for 5 h to obtain BG

nanoparticles.

The surface of BG nanoparticles was functionalized with

amine groups using APTES. BG nanoparticles of 0.1 g were

added to 50 ml toluene and sonicated for 30 min to a homoge-

neous solution. One milliliter of APTES was added to this

solution and then refluxed at 80 �C for 24 h, which was followed

by a centrifugation at 10 000 rpm for 5 min and stringent

washing with toluene and ethanol. The product was dried in an

oven at 80 �C for 24 h.

The morphology of the BG nanoparticles was observed by

transmission electron microscopy (TEM). The chemical bond

structure of the nanoparticles was analyzed by Fourier transform

infrared (FT-IR; Varian 640-IR). The phase was characterized

with X-ray diffraction (XRD; Ultima IV, Rigaku). The surface

electrical potential of the nanoparticles was analyzed by a zeta

(z)-potential measurement (Zetasizer Nano, Malvern, UK) at

25 �C. The instrument determines the electrophoretic mobility of

the particles automatically and converts it to the z-potential

using a Smoluchowski’s equation.

4.3. Suspensions and EPD process

For the success of the EPD process, it is essential to prepare a

stable suspension. CH dissolved in a 1% acetic acid solution was

dispersed at 1 g l�1 in an ethanol–water co-solvent (25% v/v

water). Within the CH solution, aminated BG nanoparticles were

dispersed by ultrasonification for 30 min at varying concentra-

tions; 5, 10, 15 and 20 wt%. The homogeneous dispersion of the

BG nanoparticles within CH solution was confirmed by means of

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a turbidity test (Smart Scientific analysis, Turbiscan, Korea).

Optical transmission % of the solution was monitored every 1 h

for up to 24 h. Moreover, the coating solution was observed by

TEM by dropping the solution onto a copper grid and then

drying.

Since the particles and molecules are positively charged, the

cathode acted as a substrate for the EPD process. The Ti

substrate was placed on the cathode and the cathode–anode

distance was maintained at 11 mm. After degassing treatment in

an ultrasonic bath, a DC voltage was applied using a powder

supply (N5771A, 300V/5A; Agilent Technologies). The EPD

coating process was optimized by varying parameters including

the pH of the suspension and coating, voltage, and time. The pH

of the composite suspensions was varied (3.1–3.6) using acetic

acid and sodium hydroxide solutions, an appropriate pH range

for the EPD of the composite solutions, as the EPD above pH 3.6

resulted in an inhomogeneous coating morphology. A DC

voltage was varied from 20–80 V, and the deposition time up to 8

min. EPD coating was done at ambient conditions, and after

coating the samples were taken, washed gently and dried for

further tests. The coating samples containing 5, 10, 15 and 20

wt% BG nanoparticles were designated as 5BGn, 10BGn, 15BGn

and 20BGn, respectively.

4.4. Characterizations of coatings

The microstructure of the EPD processed samples was charac-

terized by scanning electron microscopy (SEM) using a S-3000H

microscope (Hitachi, Japan). The cross-sectional images of

SEM taken from 3–5 samples for each composition were

observed for the approximation of coating thickness. Ther-

mogravimetric analysis (TGA; TGA N-1500; Scinco, South

Korea) of the deposits was carried out using a portion of the

coating layer scraped from the Ti substrate under operation at

temperature up to 900 �C at a heating rate of 10 �C min�1.

Based on this, the quantity of BG nanoparticles in the

composite coatings was deduced. The crystal phase and chem-

ical bond status of the samples were characterized by XRD and

FT-IR, respectively.

The apatite forming ability of the coating layers was investi-

gated in 2� simulated body fluid (SBF) with ionic concentrations

2 times higher than SBF (Na+, K+, Mg2+, Ca2+, Cl�, HCO3�,

HPO42� and SO4

2� were 284.0, 10.0, 3.0, 5.0, 295.6, 8.4, 2.0 and

1.0, respectively). The 2� SBF accelerates the apatite induction

process and thus has generally been used to test the apatite

forming ability of bioactive materials within a shorter period.65,66

Each coated sample (dimension of 10 mm � 10 mm � 2 m) was

contained in 10 ml of 2� SBF and then incubated at 37 �C for

different periods (1, 3, 5, 7, 10, 14, 21 and 28 days). At each time,

the sample were removed, washed with distilled and deionized

water and dried. The change in the surface morphology and

chemical bond structure of the samples was characterized with

SEM and FT-IR, respectively. The weight change of the samples

according to the apatite formation was also recorded. Three

replicate samples were used for each condition and averaged.

The degradation of the coating layer was studied in phosphate

buffered saline (PBS, pH 7.4).67–69 Each sample (dimension of 10

mm� 10 mm� 2 m) immersed in 30 ml PBS at 37 �C for various

periods (7, 21, 35 and 50 days) was taken out and the weight

24954 | J. Mater. Chem., 2012, 22, 24945–24956

change was recorded. Three replicate samples were used for each

condition and averaged.

4.5. Cellular study: proliferation and osteoblastic

differentiation

The effects of the coated samples on the in vitro cell growth and

osteogenic differentiation were examined. For the cell tests, a

composite composition containing 10% BG nanoparticles was

used as the representative. Each sample (Ti control, CH coating,

‘CH’, and CH–10% BG nanoparticle coating, ‘10BGn’) sterilized

with 70% ethanol was placed in each well of 24-well plates. Pre-

osteoblastic cells (MC3T3-E1; American Type Culture Collec-

tion (ATCC), USA) were plated at 2� 104 cells onto each sample

and then cultured in a-minimal essential medium (a-MEM;

Gibco, USA) supplemented with 10% fetal bovine serum (FBS;

Gibco) containing 1% penicillin–streptomycin under 5% CO2/

95% air at 37 �C. After culturing for 1, 3 and 7 days, the cell

proliferation level was assessed by the cell counting kit assay

(CCK-8, Dojindo, Japan). The cell morphology on the samples

was also observed by SEM after fixing the cells in 2.5% glutar-

aldehyde, dehydrating them with ascending concentrations of

ethanol (50, 70, 90 and 100%) and coating with gold.

Osteogenic differentiation of cells on the coating samples was

assessed by means of expression of genes related to bone

including collagen type I (Col I), alkaline phosphatase (ALP),

bone sialoprotein (BSP), osteopontin (OPN) and osteocalcin

(OCN). After culture for 7 and 14 days, total RNA was extracted

from the cells using a RNeasy Mini kit (Qiagen, South Korea). A

2 mg of the total RNA were used to perform the reverse tran-

scriptase (RT) reaction. The real-time polymerase chain reaction

(PCR) was conducted using a SYBR Green PCR kit (Quantace,

GCbiotech, Netherlands) in a Rotor-Gene 3000 spectrofluoro-

metric thermal cycler (Corbett Research, Australia). After the

real-time PCR run, the Ct value was used to determine the effi-

ciency of different genes relative to b-actin, which was used as an

internal control (DCt ¼ Ct gene � Ct b-actin). The mRNA in

each sample was then calculated by the comparative DDCt (DCt

gene � DCt control) value method. The sense and antisense

primers were designed according to published cDNA sequences

available in GenBank. Each measurement was performed in

triplicate (n ¼ 3).

4.6. Drug delivery study: incorporation, release and

antibacterial effects

Na–ampicillin was used as a model drug for the loading and

release tests of drug from the EPD coatings. Coating suspension

of either pure chitosan or chitosan–10% BG nanoparticles was

prepared in 1% acetic acid/distilled water. The amount of chi-

tosan was fixed at 100 mg. Within the suspension, ampicillin was

dissolved at two different amounts (5 mg ‘low’ and 10 mg ‘high’),

and the EPD process was carried out at 40 kV for 5 min.

The release test of ampicillin was performed in PBS at pH 7.4

and 37 �C. Each sample was incubated in PBS for different times

up to 10–11 weeks. At each time point, the sample was taken out

and the solution containing eluted ampicillin was assessed by

UV-vis spectroscopy using a Libra S22 apparatus (Biochrom,

UK) by monitoring the changes in the absorbance at a

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characteristic wavelength, 230 nm. A series of standard Na–

ampicillin solutions in deionized water (10–100 mg ml�1) were

prepared to obtain a linear calibration curve (r2 ¼ 0.99) that

obeys Beer’s law A ¼ abc, where A is the absorbance, a is a

constant known as absorbtivity coefficient, c is the concentra-

tion, and b is the cell bath length, which is constant. To eliminate

any possible interference of the degraded products, blank solu-

tions for the UV-spec. assay was prepared, by collecting solu-

tions from the coatings free of ampicillin at the same incubation

time as the drug-eluting period.

Antibacterial effects of the ampicillin released from the coat-

ings were investigated by means of an agar diffusion test against

Streptococcus mutants (from ATCC, USA). Coated samples with

or without ampicillin were used. After spreading 100 ml aliquot

of Streptococcus mutants directly onto the agar plate and incu-

bated overnight at 37 �C, each sample was placed onto the agar

plate, and the inhibitory zone formed by the released ampicillin

from the coating layer was visualized during periods for up to 5

days with 24 h interval.

4.7. Statistics

Data are presented as the mean � standard deviation and the

differences between groups were compared using a Student’s

t-test. Statistical significance was considered at p < 0.05 and

p < 0.01.

Acknowledgements

This work was supported by the Priority Research Centers

Program (no. 2009-0093829) and WCU program (no. R31-

10069) through the NSF, funded by the MEST, Republic of

Korea. Authors also thank the assistance of Mrs Hwang KH in

the cellular assays.

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