materialsinmedicine2013 sandra alcaraz
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In vitro bioactivity, cytocompatibility, and antibiotic releaseprofile of gentamicin sulfate-loaded borate bioactive glass/chitosan
composites
Xu Cui Yifei Gu Le Li Hui Wang
Zhongping Xie Shihua Luo Nai Zhou
Wenhai Huang Mohamed N. Rahaman
Received: 4 January 2013 / Accepted: 17 June 2013/ Published online: 3 July 2013
Springer Science+Business Media New York 2013
Abstract Borate bioactive glass-based composites have
been attracting interest recently as an osteoconductive car-rier material for local antibiotic delivery. In the present
study, composites composed of borate bioactive glass par-
ticles bonded with a chitosan matrix were prepared and
evaluated in vitro as a carrier for gentamicin sulfate. The
bioactivity, degradation, drug release profile, and compres-
sive strength of the composite carrier system were studied as
a function of immersion time in phosphate-buffered saline at
37 C. The cytocompatibility of the gentamicin sulfate-
loaded composite carrier was evaluated using assays of cell
proliferation and alkaline phosphatase activity of osteogenic
MC3T3-E1 cells. Sustained release of gentamicin sulfate
occurred over *28 days in PBS, while the bioactive glass
converted continuously to hydroxyapatite. The compressive
strength of the composite loaded with gentamicin sulfate
decreased from the as-fabricated value of 24 3 MPa to
*8 MPa after immersion for 14 days in PBS. Extracts of
the soluble ionic products of the borate glass/chitosan
composites enhanced the proliferation and alkaline phos-
phatase activity of MC3T3-E1 cells. These results indicate
that the gentamicin sulfate-loaded composite composed of
chitosan-bonded borate bioactive glass particles could be
useful clinically as an osteoconductive carrier material for
treating bone infection.
1 Introduction
Bacterial infection is a serious complication in orthopedic
surgery. The reported rate of infection varies from *1 %
for total joint replacement to *23 % for open factures.
The incidence of bacterial infection is expected to increase
with the increasing use of orthopedic fixation devices and
total joint replacement surgeries [1]. Current treatments
such as prolonged systemic antibiotic therapy and surgical
intervention have limitations [2, 3]. In systemic antibiotic
therapy, the drug concentration in the blood can often
increase rapidly up to a peak value soon after administra-
tion and then decrease rapidly thereafter. As a result, some
drugs can be toxic at their peak values and ineffective
below a threshold level in the blood [4]. Surgical removal
of infected implants that are secured to bone results in
skeletal deficiency and attendant difficulty in subsequent
reconstruction. The current care for infected joint implants
involves prolonged antibiotic delivery, two major opera-
tions, and morbidity.
Local delivery of high doses of antibiotics is desirable in
treating bone infections [5]. High local concentrations of
antibiotics can facilitate the delivery of antibiotics by dif-
fusion to avascular areas that are inaccessible by systemic
antibiotic. In some cases, infecting organisms that are
resistant to drug concentrations achieved by systemic
antibiotics are susceptible to the higher drug concentrations
provided by local antibiotic delivery. An ideal system for
local delivery of antibiotics should provide controlled
delivery of higher concentrations of antibiotics to the site
of infection and yet minimize the risks of systemic toxicity
X. Cui
Y. Gu
L. Li
H. Wang
N. Zhou
W. Huang (&)Institute of Bioengineering and Information Technology
Materials, Tongji University, Shanghai 200092, China
e-mail: [email protected]; [email protected]
Z. Xie S. Luo
Department of Orthopaedic Surgery, Shanghai Sixth Peoples
Hospital, Jiaotong University, Shanghai 200233, China
M. N. Rahaman
Department of Materials Science and Engineering, Center for
Bone and Tissue Repair and Regeneration, Missouri University
of Science and Technology, Rolla, MO 65409, USA
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associated with traditional methods of intravenous deliv-
ery. The delivery system should be bioresorbable or bio-
degradable to avoid the need for a second operation to
remove a non-degradable carrier. At the same time, the
delivery system should also provide a matrix for supporting
bone regeneration [6]. This is particularly advantageous in
osteomyelitis associated with infected prosthetic implants
in which bone loss is inevitable, when well-fixed, infectedmetal implants are removed.
Two widely used methods for local delivery of antibi-
otics involve antibiotic-loaded poly(methyl methacrylate)
(PMMA) cement and collagen sponge. However, PMMA is
not biodegradable and it can present a surface on which
secondary bacterial infection can occur. PMMA spacers
used to treat deep implant infections must be removed after
resolution of the infection, which requires a second sur-
gery. Collagen sponge is biodegradable and has other
advantages over PMMA cement. However, concerns have
been expressed about the suitability of collagen sponge
because of its reported rapid antibiotic release rate [7].Synthetic biodegradable polymers such as poly(lactic acid)
(PLA), poly(glycolic acid) (PGA), and their copolymers
(PLGA) have been studied as carrier materials because
they can provide more controllable antibiotic release rates.
However, these polymers are not bioactive, and they lack
the ability to form a strong bond to living bone [8].
As a drug delivery system, bioactive glass can provide
several attractive properties in the treatment of bone
infection [9, 10]. In vivo, bioactive glass converts to
hydroxyapatite (HA), the mineral constituent of bone, and
bonds firmly with hard and soft tissues [11,12]. Bioactive
glass heals to host bone, and is osteoconductive as well as
osteoinductive. During its conversion to HA, bioactive
glass releases ions (e.g., calcium ions) and soluble silica
that promote osteogenesis and activate osteogenic gene
expression [1315]. Bioactive glass has been successfully
applied in orthopedic surgery and dentistry, mainly for
repairing osseous, cystic, and periodontal defects, as well
as tumors and other lesions after resection in the appen-
dicular skeleton [16].
One of the more recently studied carriers is borate
bioactive glass [1, 10, 17]. Borate glass differs from the
more widely researched silicate bioactive glasses (such as
45S5 glass) in that the glass network is based on B2O3instead of SiO2. Some borate glasses show excellent bio-
active, biodegradable, and osteoconductive properties for
biomedical applications [1720], and they can degrade
more completely when compared to silicate bioactive
glasses [21, 22]. Porous three-dimensional (3D) scaffolds
of borate bioactive glasses have shown the capacity to
support the proliferation of osteogenic MLO-A5 cells
in vitro, tissue infiltration in a rat subcutaneous implanta-
tion model in vivo, and bone regeneration in rat calvarial
defects [10, 23, 24]. Those results indicated that borate
bioactive glass scaffolds could serve as a substrate for the
repair and regeneration of bone defects.
Chitosan, a polymerized D-glucosamine polysaccharide,
has been attracting increasing interest for biomedical
applications. It can serve as a carrier for drugs and anti-
biotics with the additional benefit of antibacterial and
antifungal activity [25]. Drug elution from chitosans can becontrolled by the amount of crosslinking, the size of the
implant, and the drug loading. Chitosan can also be used as
a matrix phase of composite carriers to optimize the drug
elution characteristics of carrier materials.
The objective of this study was to create composites of
borate bioactive glass particles bonded with a chitosan
matrix and to evaluate their ability to serve as a carrier for
controlled delivery of gentamicin sulfate, a wide spectrum
antibiotic used in the treatment of osteomyelitis [26, 27].
The composite carrier combined the bioactive and osteo-
conductive properties of the borate glass phase for bone
regeneration with the drug eluting properties of the chito-san matrix for sustained release of the antibiotic for curing
bone infection. The bioactivity, cytocompatibility, com-
pressive strength, and drug release profile of the composite
carriers were investigated in vitro.
2 Materials and methods
2.1 Fabrication of borate bioactive glass/chitosan
composites
Borate bioactive glass (BG) was prepared by mixing the
required amounts of Na2CO3, K2CO3, MgCO3, CaCO3,
H3BO3, and NaH2PO4 (analytical grade; Sinopharm
Chemical Reagent Co., Ltd. China), and melting the mix-
ture in a platinum crucible for *2 h at 1,250 C. The melt
was quenched between cold steel plates, and the resulting
glass frits were crushed, ground, and sieved to give parti-
cles of size\50 lm. The composition of the borate BG (in
mol%) was 6Na2O8K2O8MgO22CaO54B2O32P2O5.
A setting liquid composed of chitosan (98 % deacety-
lated), citric acid, and glucose was used to bond the BG
particles to form a composite carrier material. (The ana-
lytical grade chemicals were purchased from Sinopharm
Chemical Reagent Co. Ltd., China). The ratio of chitosan,
citric acid, and glucose in the solution was 1:10:20 by
weight. The mixture was stirred continuously for 2 h at
room temperature to achieve a homogeneous solution.
In the preparation of the composite carrier, BG particles
(1 g), setting liquid (0.3 ml), and gentamicin sulfate pow-
der (64 mg) were mixed using an agate mortar and pestle to
achieve a homogeneous mixture. Then the mixture was
filled into a cylindrical polyethylene mold (4.7 mm in
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diameter 9 3.5 mm) and allowed to harden for 30 min.
The pellets were removed, and dried for 24 h at room
temperature for subsequent use. Pellets composed of BG
particles bonded with the setting liquid but without gen-
tamicin sulfate were prepared using a similar procedure,
and served as control samples.
For comparison, pellets of calcium sulfate (CaSO4)
loaded with gentamicin sulfate were also prepared andevaluated. Calcium sulfate was used because it is an
inorganic carrier that is used clinically. The amount of
gentamicin sulfate loaded into the calcium sulfate was the
same as that for the BG/chitosan composites, and the
gentamicin sulfate-loaded calcium sulfate pellets were
prepared by using a solution of sodium chloride as the
setting liquid.
2.2 Degradation and bioactivity of bioactive glass/
chitosan composites
The degradation of the BG/chitosan composites with andwithout gentamicin sulfate was studied by measuring the
weight loss of the pellets and the pH of the solution as a
function of immersion time (up to 90 days) in phosphate-
buffered saline (PBS). Four pellets from each group were
together immersed in 10 ml PBS (starting pH = 7.27.4;
PO43- concentration = 0.06 M) in sterile polyethylene
containers at 37 C. The weight loss was measured after
removing the pellets from the PBS, washing them with
deionized water, and drying at 90 C, while the pH of the
solution was measured using a pH meter (FE20; Mettler
Toledo). The boron concentration in the PBS, resulting
from the degradation of the BG in the composite, was
determined using inductively-coupled plasma atomic
emission spectroscopy (ICP-AES; Optima 2100 DV;
USA). Four samples in each group were measured at each
immersion time, and the results were expressed as an
average standard deviation (sd).
The morphology and microstructure of the BG/chitosan
composites with and without gentamicin sulfate were
examined before and after immersion in PBS using field
emission scanning electron microscopy (FESEM) (Hitachi
S-4700; Tokyo, Japan). The samples were sputter-coated
with gold, and examined at an accelerating voltage of
20 kV and a working distance of 13.3 mm. The presence of
crystalline phases in the BG/chitosan samples was studied
by X-ray diffraction (XRD) (D/max-2500VB2?/PC; Rig-
aku, Tokyo, Japan) using graphite monochromatized Cu
Ka radiation (k = 0.15406 nm) at a scan rate of 10/min,
in the range 1080 2h. Compositional analysis of the
composites was performed using Fourier transform infrared
(FTIR) spectroscopy (Bruker EQUINOX SS/HYPE-
RION2000; Germany). FTIR was performed in the wave-
number range 4004,000 cm-1, on disks prepared from a
mixture of 2 mg of the composite powder and 150 mg of
high-purity KBr. Each sample was scanned 32 times at a
scan rate of 0.04 cm-1.
2.3 Compressive strength of carrier materials
The compressive strength of the BG/chitosan composites
with and without gentamicin sulfate and the calcium sulfatewith gentamicin sulfate was measured as a function of
immersion time in PBS. Cylindrical samples (5 mm in
diameter 9 10 mm) were tested at a crosshead speed of
0.5 mm/min in a mechanical testing machine (CMT6104;
SANS Test Machine Inc., China). For each measurement,
five pellets from each group were together immersed in
12.5 ml PBS) at 37 C and tested. The compressive
strength at each immersion time was determined as an
average sd.
2.4 Release of gentamicin sulfate in PBS
Four pellets each of the as-prepared BG/chitosan com-
posite with gentamicin sulfate and the calcium sulfate with
gentamicin sulfate were weighed, and the amount of gen-
tamicin sulfate in each pellet was determined from the
mass of the pellet and the composition of the starting
mixture. Then the four pellets from each group were
immersed in 10 ml PBS at 37 C. At selected immersion
times, the eluting solution was removed, stored at -20 C,
and assayed within 7 days using high performance liquid
chromatography (HPLC). The amount of gentamicin sul-
fate released from the four pellets in each group at each
time point was expressed as an average sd.
2.5 In vitro antibacterial activity assay
S. aureus is a Gram-positive bacterium that can cause a
wide range of suppurative infections [28]. In this study, S.
aureus was used as a model bacterium to evaluate the
antibacterial activity of gentamicin sulfate loaded BG/
chitosan composite, and the BG/chitosan composite with-
out gentamicin sulfate was used as the blank controller.
For qualitative analysis, inhibition zone of the S. aureus
cultured on the LB agar plates was assessed [29]. Briefly,
S. aureus suspension (100 ml) was first spread onto the
agar plates and the BG/chitosan composite with a diameter
of 4.7 mm and a mass of around 80 mg were pasted onto
the agar plate. All the samples were sterilized for 3 h
before they were pasted. The amount of gentamicin sulfate
in BG/chitosan composite was approximately 5 mg. The
bacteria were then incubated at 37 C for 24 h and the
inhibition zone for each sample on the plate was visually
inspected. The inner and outer diameters and calculated
diameter difference were measured.
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2.6 Cell culture
The osteogenic MC3T3-E1 cell line used in these experi-
ments was provided by Shanghai 6th Hospital, China. The
cells were cultured in a-MEM supplemented with 10 %
fetal bovine serum (FBS) plus 100 U/ml penicillin and
100 lg/ml streptomycin sulfate. Once 8090 % confluence
was reached, and after they were trypsinized in 0.25 %pancreatic enzymes, cells of generation 515 were used in
cytotoxicity studies. All cell cultures were performed at
37 C in a humidified atmosphere of 5 % CO2.
Upon immersion of the BG/chitosan composite carrier
in an aqueous solution, the borate BG particles can
degrade, releasing boron, presumably as borate (BO3)3-
ions, and other ions, such as Na? and K? into the solution.
The effect of the soluble ionic product of the BG/chitosan
composite degradation on the proliferation of the MC3T3-
E1 cells was evaluated using assays of MTT hydrolysis.
Pellets of BG/chitosan composites with and without gen-
tamicin sulfate were immersed in culture media for 24 and48 h, using a fixed ratio of the BG/chitosan surface area to
the culture media volume (3 cm2/ml). At each immersion
time, extracts were removed from the media, sterilized by
filtering through a 0.22 lm filter, and diluted for use in
subsequent cell culture. Extracts of culture media without
the glass dissolution product were used as the control.
MC3T3-E1 cells were seeded in 96-well plates a density
of 5 9 105 cells per well in 1 ml of complete medium. The
cells were incubated in a humidified incubator at 37 C and
5 % CO2 atmosphere. After the cells were seeded for 4 h,
the medium was replaced with low-serum, fresh a-MEM
(0.5 % FBS) and synchronized for 812 h. Then extracts of
the 24-h dissolution product of the BG/chitosan composites
described above were added to one group of cells, while
the 48-h dissolution product was added to another group of
cells. In the control group, media composed of 90 %
DMEM (Gibco-BRL; USA), 10 % FBS, 100 IU/ml pen-
ethamate, and 100lg/ml streptomycin (all purchased from
Gibco, USA) was incubated for 24 h at 37 C i n a
humidified atmosphere of 5 % CO2, then stored at 4 C
prior to use. Twelve parallel wells were used for each
group. After incubation for a further 24 h, 20 ll of MTT
(methyl thiazolyl tetrazolium,) solution was added to each
well. After incubation for 4 h, the culture medium was
removed and dimethylsulfoxide (DMSO; Sigma, USA) was
added to dissolve the formazan and the system was mixed
thoroughly. After adjusting to zero with the control group,
the optical density (OD) was measured at a wavelength of
490 nm in a BMG FLUOstar Optima plate reader.
After the culture medium was removed, the remaining
cells were collected and lysed with 0.2 % Triton X-100.
Then the lysate was centrifuged for 5 min, and the super-
natant was collected for measurement of alkaline
phosphatase (ALP) activity. ALP activity was evaluated
using a colorimetric assay (Alkaline Phosphatase Liqui-
Color; Stanbio, Boerne, TX, USA) based on the hydrolysis
of 4-nitrophenyl phosphate to 4-nitrophenol, formation of
which is directly proportional to ALP activity. Absorbance
was determined at 405 nm using a microplate reader (DTX
800 Multimode Detector, Beckman Coulter, Brea, CA,
USA). Data were normalized to the total cell protein, asmeasured using a commercial kit (DC Protein Assay Kit,
Bio-Rad, Hercules, CA, USA) and are expressed as
micromoles of 4-nitrophenol produced per hour per milli-
gram of protein (lmol h-1 mg-1).
2.7 Evaluation of cell adhesion
MC3T3-E1 cells suspended in 5 ml medium (19 104
fibroblasts/ml; 1 9 105 macrophages/ml) were placed on
discs of the gentamicin sulfate-loaded BG/chitosan com-
posite. After incubation for 7 days, the samples were
placed in an Eppendorf vessel and fixed overnight in 2.5 %glutaraldehyde in 0.1 M sodium cacodylate buffer (pH
7.2). After washing in the buffer and post-fixation in 1 %
buffered osmium tetroxide for 1 h, the samples were
dehydrated in a graded series of ethanol and embedded in
Epon-araldite resin. Examination of the cell morphology
was performed using SEM.
2.8 Statistical analysis
All data for the cell culture experiments (n = 4 or 5) are
presented as a mean SD. Analysis for differences
between groups was performed using one-way ANOVA.
Differences were considered significant forP\ 0.05.
3 Results
3.1 Biodegradation of BG/chitosan composites
When immersed in an aqueous solution containing phos-
phate ions, borate BG degrades, releasing boron and the
glass network modifiers (such as Na? and K?) into the
solution. At the same time, Ca2? ions released from the
glass react with phosphate ions to form an amorphous
calcium phosphate (ACP) or HA-like material on the glass
[21,22]. The conversion of the glass to ACP or HA results
in a weight loss. Consequently, the weight loss of the glass
or the concentration of boron released into the solution can
be used to study the degradation of borate BG.
Figure1 shows the cumulative amount of boron
released into PBS as a function of immersion time of the
BG/chitosan composites with and without gentamicin sul-
fate. The boron concentration increased rapidly during the
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first 45 days, but increased more slowly thereafter. Based
on the total amount of boron present in the as-prepared BG/
chitosan composites, *23 % of the boron initially present
in the BG was released after 1 day for the BG/chitosan
composite with gentamicin sulfate. In comparison, *33 %
of the boron was released from the BG/chitosan composite
without gentamicin sulfate after 1 day. After an immersion
time of 4 days, the boron concentration released into thesolution was 29 and 40 %, respectively, for the BG/chito-
san composites with and without gentamicin sulfate. After
90 days, the amount of boron released was 65 and 75 %,
respectively, for the BG/chitosan composites with and
without gentamicin sulfate. In general, the trend in the
boron release profile was similar for the BG/chitosan
composites with and without gentamicin sulfate, but the
release was slower for the composite with gentamicin
sulfate.
The fractional weight loss of the BG/chitosan compos-
ites with and without gentamicin sulfate (defined as weight
loss normalized to the original weight) (Fig. 2a) showed asimilar trend to the boron release data described above. The
weight loss increased rapidly during the first 45 days of
immersion and more slowly thereafter. The weight loss
after 710 days was *35 % for both groups of composites.
At longer times, the weight loss of the BG/chitosan com-
posite with gentamicin sulfate was higher than the com-
posite without gentamicin sulfate, but the difference was
not significant.
The pH of the PBS (starting value = 7.4) as a function
of immersion time of the BG/chitosan composites with and
without gentamicin sulfate is shown in Fig.2b. The pres-
ence of the gentamicin sulfate in the composite did not
have a significant effect on the pH of the solution. The pH
increased rapidly during the first 45 days, and after
reaching a maximum value of 8.68.7 after *10 days, it
remained nearly steady at *8.5 at longer immersion times.
When the borate BG particles alone were immersed in
PBS, the pH was observed to increase as high as 9.6 [30].
Presumably the presence the chitosan matrix in the com-
posites used in this study had an effect in reducing the pH,
which would be beneficial for reducing an inflammatoryresponse resulting from a higher alkalinity [31].
3.2 In vitro bioactivity of BG/chitosan composites
XRD patterns of the BG/chitosan composites with and
without gentamicin sulfate are shown in Fig.3 after
immersion of the composites for 20 and 90 days in PBS.
The major peaks in the patterns corresponded to those of a
reference HA, Ca10(PO4)6(OH)2 (JCPSD 72-1243), indi-
cating the formation of HA-like material by reaction of the
BG particles in the composite with PBS. The diffraction
peaks were broad and weak in intensity (height) whencompared to those of the reference HA, which may be an
indication that the as-formed HA had not fully crystallized
or that the crystallite size of the HA was in the nanometer
range, or a combination of both.
Figure4 shows FTIR spectra of the BG/chitosan com-
posites with and without gentamicin sulfate after immer-
sion of the composites for 1 and 90 days in PBS. As a
reference, the spectrum of the borate BG particles (without
the chitosan matrix) is also shown. The spectrum of the
borate BG was dominated by broad resonances at 600800
and 8001,200 cm-1, attributed to the stretching vibration
of the BO bond (BIV-O) in tetrahedral [BO4] units, and
strong resonance at 1,2001,500 cm-1 attributed to
stretching of BO bond (BIIIO) in trigonal [BO3] units.
Those resonances indicated that the glass network was
composed mainly of [BO3] and [BO4] units. In addition to
a broad resonance 3,0003,700 cm-1 corresponding to the
stretching and bending vibration mode of -OH, the spectra
of the BG/chitosan composites showed the major reso-
nances characteristic of HA. They included the m3 reso-
nance at 1,045 cm-1 and the m1 resonance 963 cm-1
corresponding to PO stretching, as well as m4 resonances
at 603 and 571 cm-1 corresponding to OPO bending.
The resonances at 1414, 1550, and 1640 cm-1 are attrib-
uted to CO in CO32-, while the resonance at 870 cm-1
was attributed to POH in HPO42-.
In combination, the resonances in the FTIR spectra
indicated the formation of a carbonate-substituted HA
resulting from the conversion of the BG phase in PBS.
Since the present experiments were carried out in air, it is
likely that CO2 from the atmosphere dissolved in the
solution, giving rise to the substitution of some CO32- in
the HA lattice. While the substitution of (PO4)3- in the HA
Fig. 1 Cumulative amount of boron released from borate bioactive
glass/chitosan (BG/chitosan) composites with and without gentamicin
sulfate (GS) as a function of immersion time of the composites in
phosphate-buffered saline (PBS)
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lattice by (CO3)2- is commonly observed, it has been
reported that (CO3)2-can also substitute for OH- when the
HA is precipitated in a solution containing (CO3)2- ions
[21, 22]. The presence of gentamycin sulfate or chitosan
was not observed in the FTIR spectra of the BG/chitosan
composites with or without gentamicin sulfate, presumably
because of the small quantity of those materials in the
composites or the masking of low-intensity resonances of
those materials by the HA resonances.
SEM images of the surface of the BG/chitosan com-
posites with and without gentamicin sulfate, as-fabricated
and after immersion for 90 days in PBS are shown in
Fig.5. While he surface of the as-fabricated composites
showed some differences in microstructure (Figs.5a, b),
the presence of the gentamicin sulfate had little effect on
the final microstructure (Figs. 5c, f). After the ninety-day
immersion, a layer of spherical particles was formed on the
surface (Figs.5e, f). EDS analysis of the composites
immersed for 90 days in PBS showed that in addition to O,
the spherical particles consisted mainly of Ca and P
(Fig.6). In addition, EDS spectra showed the presence of
small quantities of Na, K, and Mg (network modifiers in
the borate glass), presumably incorporated into the parti-
cles during the conversion process. The Ca/P atomic ratio
of the spherical particles as determined by EDS was
1.62 0.02, which is close to the value (1.67) for stoi-
chiometric HA.
Fig. 2 aWeight loss of BG/chitosan composites with and without gentamicin sulfate (GS), and b pH of the solution as a function of immersion
time of the composites in PBS
Fig. 3 XRD patterns of BG/chitosan composites with and without
gentamicin sulfate after immersion of the composites in PBS: (a, c)
composite with gentamicin sulfate (GS) after immersion for 20 and
90 days, respectively; (b, d) composite without gentamicin sulfate
after immersion for 20 and 90 days, respectively
Fig. 4 FTIR spectra of BG particles, chitosan, and BG/chitosan
composites after immersion in PBS: (a) as-received chitosan; (b) as-
prepared borate BG particles; (c,e) composite with gentamicin sulfate
(GS) after immersion for 1 and 90 days, respectively; (d,f) composite
without gentamicin sulfate after immersion for 1 and 90 days,
respectively
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3.3 Compressive strength of BG/chitosan composites
The measured compressive strength of the BG/chitosan
composites with and without gentamicin sulfate, as fabri-
cated and after immersion of the composites for up to
14 days in PBS is shown in Fig. 7. For comparison, the
data for calcium sulfate loaded with gentamicin sulfate are
also shown. The compressive strength of the calcium sul-fate decreased rapidly from the as-fabricated value of
20 2 MPa to 5 2 MPa after immersion for 2 days in
PBS. In comparison, the compressive strength of the BG/
chitosan composites with gentamicin sulfate decreased
from the as-fabricated value of 24 3 MPa to
12 3 MPa after immersion for the same time in PBS.
The BG/chitosan composites without gentamicin sulfate
had a compressive strength of 42 1 MPa as fabricated,
and 16 3 MPa after immersion for 2 days in PBS. With
an increase in immersion time, the strength of the calcium
sulfate decreased rapidly to almost zero within 8-10 days,
whereas the BG/chitosan composites with or without gen-tamicin sulfate had a strength of *8 MPa even after
immersion for a longer time (14 days) (Fig.7).
3.4 Release profile of gentamycin sulfate
Data for the cumulative amount of gentamicin sulfate
released from the BG/chitosan composite and from the
calcium sulfate into PBS are shown in Fig. 8. The release
profile from both materials showed the same general trend:
a rapid increase initially followed by a much slower
increase thereafter. However, the release from the calcium
sulfate was faster. The amount of gentamicin sulfate
released from the calcium sulfate at day 1 was *65 % of
the total amount initially loaded into the as-fabricated
material, and almost all the gentamicin sulfate was released
by day 14. In comparison, release from the BG/chitosancomposites was more sustained; *40 % of the gentamicin
sulfate was released at day 1, and release continued for as
long as 28 days. The amount of gentamycin sulfate
released was *96 % for the calcium sulfate after 14 days
and *85 % for the BG/chitosan composite after 28 days
(Fig.8).
3.5 Antibacterial activity
Figure9 shows the antibacterial activity of BG/chitosan
composite with or without gentamicin sulfate. It is saw thatboth the BG/chitosan composite with and without genta-
micin sulfate display bacterial inhibition rings, but the
inhibition zone of the S. aureus for BG/chitosan with
gentamicin sulfate was larger and more obvious comparing
with that of without gentamicin sulfate. The bacterial
inhibition of BG/chitosan composite without gentamicin
sulfate may be due to the high concentration boron release
during the early stage of incubation in LB agar plates.
Fig. 5 SEM images of the surface of BG/chitosan composites as
fabricated and after immersion for 90 days in PBS: a as-prepared
composite with gentamicin sulfate (GS); b as-prepared composite
without gentamicin sulfate; c, e composite with gentamicin sulfate
after immersion; d, f composite without gentamicin sulfate after
immersion
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3.6 Cell attachment, proliferation and alkaline
phosphatase activity
Figure10 shows the morphology of MC3T3-E1 cells on
the surface of a BG/chitosan composite loaded with
gentamicin sulfate, after immersion of the composite for
7 days in the cell suspension. The MC3T3-E1 cells
appeared to attach, spread and proliferate well on surface
of the composite. Some cells were found to be completely
Fig. 6 SEM images and
corresponding EDS spectra of
BG/chitosan composites after
immersion for 90 days in PBS:
a, b composite without
gentamicin sulfate; b, d
composite with gentamicin
sulfate
Fig. 7 Compressive strength of BG/chitosan composites with and
without gentamicin sulfate (GS) after immersion in PBS for the times
shown. For comparison, the compressive strength of calcium sulfate
loaded with gentamicin sulfate is also shown
Fig. 8 Cumulative amount of gentamycin sulfate released from BG/
chitosan composite and calcium sulfate as a function of immersion
time in PBS. The amount is shown as a percentage of the total amount
of gentamicin sulfate initially loaded into the carrier material
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covered with an HA-like layer, but the spreading of the
cells was still observable.
The results of MTT assays of MC3T3-E1 cells cultured
for 24 h in media containing extracts of the ionic dissolu-
tion products of the BG/chitosan composites with and
without gentamicin sulfate are shown in Fig.11a. The
extracts were taken after immersion of the composites for
24 and 48 h in media as described in the procedure. For
both groups of composites, the 24- and 48-h extracts both
enhanced cell proliferation when compared to the control
group (cultured without the ionic dissolution product).
However, the 24-h extract had a significantly greater effect
in enhancing cell proliferation. Figure11b shows the
results for the ALP activity of MC3T3-E1 cells cultured for
24 h in media containing extracts of the ionic dissolution
products of the chitosan/BG composites with or without
gentamicin sulfate. The trend in the ALP activity was
similar to that for the cell proliferation data described
above. Both the 24- and the 48-h extracts enhanced ALP
activity, but the 24-h extract was more effective.
4 Discussion
A benefit of the BG/chitosan composite used in the present
study is the potential for providing an osteoconductive
implant for bone regeneration coupled with the ability to
serve as a carrier for local antibiotic delivery. The results
showed that in addition to being bioactive, the BG/chitosan
composite had superior compressive strength and a more
sustained gentamicin sulfate release profile when compared
to calcium sulfate, an inorganic carrier material used
clinically. The ionic dissolution products of the BG/chito-
san composite also enhanced the proliferation and functionof osteogenic cells in vitro, showing the cytocompatibility
of the composite. These results indicate that the BG/
chitosan composite developed in this study could have
potentially application in the treatment of bone infection.
4.1 Bioactivity of BG/chitosan composites
When placed in PBS, the borate BG phase in the composite
carrier degrades, releasing ions such as Na? and K?, and B,
presumably in the form of borate (BO3)3- ions. At the
same time, the Ca2? ions released from the BG reacts with
the phosphate ions present in PBS to form an HA product
on the glass [21, 22]. Since the chitosan phase does not
degrade in PBS, the measured weight loss of the composite
and the pH of the PBS therefore result primarily from the
degradation and conversion of the BG phase in the com-
posite. The results showed that the presence of gentamicin
sulfate in the composite did not significantly influence the
weight loss of the BG and the pH of the PBS (Fig. 2), but it
reduced the amount of B released into PBS (Fig.1).
The amount of B released from the BG/chitosan com-
posites into PBS provides a suitable measure of the deg-
radation of the BG phase. The amount released from the
BG/chitosan composites with and without gentamicin sul-
fate 65 and 75 %, respectively, after 90 days in PBS,
indicating that the borate BG particles were not completely
degraded. When compared to similar borate glass particles
by themselves (i.e., without incorporation in a chitosan
matrix), the degradation rate of the BG particles in the
composite was slower [21, 22]. The presence of the
chitosan matrix served not only to control the release of
gentamicin sulfate but also to reduce the degradation rate
of the encapsulated glass particles. As a result, it might be
Fig. 9 Inhibition of bacteria (S. aureus) on agar plate after incubation
for 24 h. Spot 1 and 2 represents BG/chitosan with and without
gentamicin sulfate, respectively
Fig. 10 SEM image of MC3T3-E1 cell morphology after incubation
for 7 days on BG/chitosan composite loaded with gentamicin sulfate
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possible to better match the degradation rate of the BG
phase with the rate of new bone growth to achieve opti-
mum bone regeneration.XRD, FTIR, and EDS analyses (Figs. 3,4,6) confirmed
the bioactivity of the BG/chitosan composite. After
immersion of the composite in PBS, an HA product was
formed on the composite, as a result of the degradation of
the BG particles and reaction of the released Ca2? ions
with the phosphate ions from the PBS. The HA product
was highly porous (Fig. 5), providing for easy transport of
the ionic solution through it. When compared to the XRD
pattern of commercial HA, the major peaks of the HA
product from the BG conversion were broad and had low
intensity (height), which indicated that HA product was not
fully crystallized or that the crystallite size was in thenanometer range, as confirmed by the SEM images in
Figs. 5 and 6. These partially crystallized or nanometer
size HA particles may be beneficial for cell adhesion and
proliferation [21,22].
4.2 Mechanical properties of BG/chitosan composites
The mechanical properties of an implant are critically
important for its application to the regeneration of bone in
load-bearing sites [32]. The required mechanical properties
of implants intended for loaded bone repair are not clear,
but it is generally assumed that the implant should havemechanical properties comparable to the bone to be
replaced. As fabricated, the BG/chitosan composites with
gentamicin sulfate (*6.4 wt% based on the weight of the
BG) had a compressive strength of 24 3 MPa, which is
approximately twice the highest compressive strength
reported for human trabecular (or cancellous) bone
(strength = 212 MPa) [33]. As described previously,
calcium sulfate is used clinically as a carrier for antibiotics
and drugs. The present study showed that when loaded with
gentamicin sulfate, the strength of the as-fabricated cal-
cium sulfate (20 2 MPa), was not very different from
the value given above for the as-fabricated BG/chitosancomposite. However, a major difference between the two
carrier materials was the decrease in strength as a function
of immersion time in PBS (Fig.7). While the strength of
both carriers decreased with immersion time, the strength
of the calcium sulfate carrier decreased more rapidly. The
gentamicin sulfate-loaded calcium sulfate had little
strength after immersion for 810 days. In comparison,
after immersion for a longer time (14 days), the gentamicin
sulfate-loaded BG/chitosan composite had a strength of
*8 MPa, in the range of strengths reported for human
trabecular bone (212 MPa). The data indicate that the
gentamicin sulfate-loaded BG/chitosan composites couldprovide a suitable carrier for treating infection even when
some load-bearing capability is required [34].
4.3 Gentamicin sulfate release profile and Antibacterial
activity
Release of gentamicin sulfate from calcium sulfate was
rapid with almost 100 % of the antibiotic released within
1014 days (Fig.8). This is compatible with the results of
previous studies which showed rapid elution of antibiotics
from calcium sulfate and high dissolution rate of calcium
sulfate [35]. In comparison, the BG/chitosan compositedeveloped in this study showed a more sustained release
which might be attributed to the presence of the chitosan
matrix. The free amine groups in the cellulose-like polymer
provide reactive cationic groups that can bond with anionic
groups [36]. Upon immersion in PBS, reaction between the
amine groups of chitosan and phosphate (PO4)3- ions in
PBS could lead to crosslinking of the chitosan, which could
result in a lower but more sustained antibiotic release
[17,37]. It is possible that deposition of HA on the surface
Fig. 11 Effect of BG/chitosan dissolution product on a cell prolif-
eration and b alkaline phosphatase activity of MC3T3-E1 cells.
Extracts of the dissolution product, taken after immersing the BG/
chitosan composites with and without gentamicin sulfate for 24 and
48 h in media, were added to the cell culture and incubated for 24 h.
The control group was incubated without the dissolution product of
the composites (Mean SD; n = 3. * Significantly different from
control group, P\0.05)
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of composite carrier, as observed in the present study,
could also lead to a reduction in the antibiotic release rate.
By encapsulating into the BG/chitosan composite, the
antibacterial activity of gentamicin sulfate was not been
eliminated. Comparing with the blank controller (Fig.9),
the BG/chitosan composite with gentamicin sulfate had a
more remarkable antibacterial property. It is worth to note
that even without gentamicin sulfate, the BG/chitosancomposite also showed sort of antibacterial activity. This
may be due to the high concentration of boron release into
the LB agar after incubation, and a high concentration of
boron in LB agar plate can cause the toxicity, which can
sterilizing bacteriostasis [38, 39]. But while implanted
in vivo, the dynamic body fluid environment can alleviate
or even eliminate the antibacterial activity of the high
boron concentration, then, gentamicin sulfate is the only
antibiont.
4.4 Cytocompatibility of BG/chitosan composites
When placed in an aqueous phosphate solution such as the
body fluid, borate BG degrades and converts to HA,
releasing B, presumably as borate (BO3)3- ions, and other
ions (e.g., Na?; K?) in the process. While low concentra-
tions of B are reported to be beneficial for healthy bone
growth, concerns have been expressed about the toxicity of
high B concentration. Boron is an essential element for
plants and mammals, and it reported that humans and
animals have a homeostatic control for maintaining boron
concentrations within certain limits [38, 39]. Several
studies have shown that B has a beneficial effect on the
formation, composition, and physical characteristics of
bone, and its interaction with other nutrients [39]. Sup-
plemental B in the form of boric acid has been shown to
enhance bone structure and strength in rats [40]. As a result
of its effect on the presence or activity of osteoblasts and/or
osteoclasts, boron is beneficial for bone growth and
maintenance. Boron deprivation in animals leads to
impaired growth and abnormal bone development [4045].
The effect of varying the B2O3content of borate BGs on
their ability to support the proliferation of osteogenic
MC3T3-E1 cells in vitro has been studied recently [41]. It
was found that below a certain threshold level, the B2O3content of the glass had little effect on cell proliferation. In
comparison, above the threshold level, cell proliferation
decreased markedly with increasing B2O3 content of the
glass. An investigation of the effects of boron concentration
(0, 1, 10, 100, and 1,000 ng/ml) on the osteogenic differen-
tiation of human bone marrow stromal cells (BMSCs)
showed that the proliferation of BMSCs was not different
from the control group for boron additions of 1, 10, and
100 ng/ml [39]; however, a boron concentrationof 1,000 ng/
ml inhibited the proliferation of BMSCs at days 4, 7, and 14.
In the present study, the effect of the ionic dissolution
products of the BG/chitosan composites on the attachment,
proliferation and ALP activity of MC3T3-E1 cells was
studied to evaluate the cytocompatibility of the composite
carrier. After an incubation time of 7 days, the MC3T3-E1
cells were found to attach, spread, and proliferate on the
composites (Fig.9). Assays of MTT hydrolysis and ALP
activity showed that extracts of the BG/chitosan dissolutionproduct, taken 24 and 48 h after immersing the composite
in culture medium, enhanced the proliferation and ALP
activity of MC3T3-E1 cells. However, the 24-h extract
showed a significantly better capacity to enhance cell
proliferation and ALP activity. Since the 48-h extract had a
higher B concentration, the results of the MTT and ALP
assays showed that the ionic dissolution product enhanced
MC3T3-E1 cell proliferation and ALP activity in a dose-
dependent manner. The 24-h extract with a lower B con-
centration had a better capacity to enhance cell prolifera-
tion and ALP activity than the 48-h extract with a higher B
content.In vivo, B is excreted primarily in the urine regardless of
the method of administration. In humans, 99 % of a single
intravenous dose of boric acid was excreted in the urine
over a period of 120 h; the half-life was determined to be
2124 h [4045]. The dynamic in vivo environment can
lead to excretion of B resulting from the degradation of the
BG phase in the BG/chitosan composite, thereby diluting
the B concentration. In our current studies, borate BG/
chitosan composites loaded with gentamicin sulfate are
being used to treat osteomyelitis in animal models and the
results of those experiments will be reported in a sub-
sequent publication.
5 Conclusions
Composites of borate bioactive glass particles bonded with a
chitosan matrix (BG/chitosan composites) showed the
ability to provide sustained release of the antibiotic genta-
micin sulfate in PBS. Release of the antibiotic occurred over
a period of *28 days, when *84 % of the gentamicin
sulfate initially loaded into the composite was released. In
comparison, calcium sulfate loaded with the same amount of
gentamicin sulfate released almost 100 % of the antibiotic
within *14 days. The gentamicin sulfate-loaded BG/
chitosan composite (as-fabricated compressive strength =
24 3 MPa) retained a strength of*8 MPa after immer-
sion for 14 days in PBS, whereas the calcium sulfate (as
fabricated strength = 20 2 MPa) lost its strength almost
completely within 810 days. The BG particles converted to
hydroxyapatite, showing the bioactivity of the composite
carrier. Ionic dissolution products of the BG/chitosan com-
posite enhanced the cell proliferation and alkaline
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phosphatase activity of osteogenic MC3T3-E1 cells, show-
ing the cytocompatibility of the composite. Together, the
results indicate that the gentamicin sulfate-loaded BG/
chitosan composite developed in this study could have
potential clinical application as an osteoconductive carrier
for treating bone infection.
Acknowledgments This work was supported by the National Nat-ural Science Foundation of China through the Projects 51072133,
81000788, 81201377 and by the Shanghai Science Committee
through the project 12JC1408500.
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