MaterialsInMedicine2013 Sandra Alcaraz

8/22/2019 MaterialsInMedicine2013 Sandra Alcaraz

1/13

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

1 3

J Mater Sci: Mater Med (2013) 24:23912403

DOI 10.1007/s10856-013-4996-0


2/13

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

2392 J Mater Sci: Mater Med (2013) 24:23912403

1 3


3/13

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.

J Mater Sci: Mater Med (2013) 24:23912403 2393

1 3


4/13

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


1 3


5/13

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)


1 3


6/13

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


1 3


7/13

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


1 3


8/13

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


1 3


9/13

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


1 3


10/13

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)


1 3


11/13

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


1 3


12/13

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.

References

1. Xie ZP, Liu X, Huang WH, et al. Treatment of osteomyelitis and

repair of bone defect by degradable bioactive borate. J Controlled

Release. 2009;139:11826.

2. Bucholz HW, Heinet K, Foerster GW. Infected prostheses: the role

of antibiotic cement. In: DAmbrosia RD, Marier RL, editors.

Orthopaedic infections. New Jersey: Slack Inc; 1989. p. 47788.

3. Zhao L, Yan X, Yu C, et al. Mesoporous bioactive glasses for con-

trolled drug release. Microporous Mesoporous Mater. 2008;109:

2105.

4. Xue JM, Shi M. PLGA/Mesoporous silica hybrid structure for

controlled drug release. J Controlled Release. 2004;98:20917.

5. Xia W, Chang J. Well-ordered mesoporous bioactive glasses

(MBG): a promising bioactive drug delivery system. J Controlled

Release. 2006;110:52230.

6. Jain AK, Panchagnula R. Skeletal drug delivery systems. Int J

Pharm. 2000;206:112.

7. Lee CH, Singla A, Lee Y. Biomedical applications of collagen.

Int J Pharm. 2001;221:122.

8. Saito K, Hoshino T. Apatite-coated solid composition. US Patent

6344209, 2002.

9. Liu X, Xie ZP, Huang WH, et al. Bioactive borate glass scaffolds:

in vitro and in vivo evaluation for use as a drug delivery system inthe treatment of bone infection. J Mater Sci Mater Med. 2010;

21:57582.

10. Zhang X, Jia WJ, Huang WH, et al. Teicoplanin-loaded borate

bioactive glass implants for treating chronic bone infection in a

rabbit tibia osteomyelitis model. Biomaterials. 2010;31(22):

586574.

11. Rahaman MN, Day DE, Fu Q, et al. Bioactive glass in tissue

engineering. Acta Biomater. 2011;7:235573.

12. Fu Q, Saiz E, Rahaman MN, et al. Bioactive glass scaffolds for

bone tissue engineering: state of the art and future perspectives.

Mater Sci Eng C. 2011;31:124556.

13. Rahaman MN, Brown RF, Day DE, et al. Bioactive glasses for non-

bearing applications in total joint replacement. Semin Arthroplasty.

2007;17:10212.

14. Liang W, Rahaman MN, Day DE, et al. Bioactive borate glassscaffold for bone tissue engineering. J Non-Cryst Solids. 2008;

354:16906.

15. Huang TS, Rahaman MN, Day ED, et al. Porous and strong

bioactive glass (1393) scaffolds fabricated by freeze extrusion

technique. Mater Sci Eng C. 2011;31:14829.

16. Domingues ZR, Cortes ME, Gomes TA, et al. Bioactive glass as a

drug delivery system of tetracycline and tetracycline associated

with b-cyclodextrin. Biomaterials. 2004;25:32733.

17. Jia WT, Zhang X, Huang WH, et al. Novel borate glass chitosan

composite as a delivery vehicle for teicoplanin in the treatment of

chronic osteomyelitis. Acta Biomater. 2010;6:8129.

18. Yao AH, Wang D, Huang WH, et al. In vitro bioactive charac-

teristics of borate-based glasses with controllable degradation

behavior. J Am Ceram Soc. 2007;90(1):3036.

19. Liu X, Huang WH, Fu HL, et al. Bioactive borosilicate glass

scaffolds: in vitro degradation and bioactivity behaviors. J Mater

Sci Mater Med. 2009;20:123743.

20. Brown RF, Day DE, Rahaman MN, et al. Growth and differen-

tiation of osteoblastic cells on 1393 bioactive glass fibers and

scaffolds. Acta Biomater. 2008;4:38796.

21. Huang WH, Rahaman MN, Day DE, et al. Mechanisms for

converting bioactive silicate, borate, and borosilicate glasses to

hydroxyapatite in dilute phosphate solutions. Phys Chem Glasses.

2006;47(6):64758.

22. Huang WH, Day DE, Rahaman MN, et al. Kinetics and mecha-

nisms of the conversion of silicate (45S5), borate, and borosili-

cate glasses to hydroxyapatite in dilute phosphate solutions.

J Mater Sci Mater Med. 2006;17:58396.

23. Fu Q, Rahaman MN, Fu HL, et al. Silicate, borosilicate, and borate

bioactive glass scaffolds with controllable degradation rate for

bone tissue engineering applications. I. Preparation and in vitro

degradation. J Biomed Mater Res Part A. 2011;95(1):16471.

24. Fu Q, Rahaman MN, BalBS, et al.Silicate, borosilicate,and borate

bioactive glass scaffolds with controllable degradation rate for

bone tissue engineering applications. II. In vitro and in vivo bio-

logical evaluation. J Biomed Mater Res Part A. 2011;95(1):1729.

25. Majeti NV, Ravi K. A review of chitin and chitosan applications.

React Funct Polym. 2000;46:127.

26. Frutos P, Diez-Pena E, Frutos G, et al. Release of gentamicin

sulphate from a modified commercial bone cement. Effect of (2-

hydroxyethyl methacrylate) comonomer and poly(N-vinyl-2-

pyrrolidone) additive on release mechanism and kinetics. Bio-

materials. 2002;23:378797.

27. Clarot I, Chaimbault P, Hasdenteufel F, et al. Determination of

gentamicin sulfate and related compounds by high-performance

liquid chromatography with evaporative light scattering detec-

tion. J Chromatogr A. 2004;1031:2817.

28. Lowy FD. Staphylococcus aureus infections. N Engl J Med.

1998;339:52032.

29. Boyle VJ, Fancher ME. Ross Jr R W. Rapid, modified Kirby-

Bauer susceptibility test with single, high-concentration antimi-

crobial disks. Antimicrob Agents Chemother. 1973;3:41824.

30. Ning J, Wang DP, Huang WH, et al. Preparation of borosilicate

glass and their bioactivity and biodegradation in vitro. J Chin

Ceram Soc. 2006;34(11):132630.

31. Zhang X, Fu HL, Huang WH, et al. In vitro bioactivity and

cytocompatibility of porous scaffolds of bioactive borosilicate

glasses. Chin Sci Bull. 2009;54(24):4638.

32. Maeda H, Ishida EH, Kasuga T. Hydrothermal preparation of to-

bermorite incorporating phosphate species. Mater Lett. 2012;68:

3824.

33. Kokubo T, KimHM, Kawashita M. Novelbioactive materials with

different mechanical properties. Biomaterials. 2003;24:216175.

34. Goldstein SA. The mechanical properties of trabecular bone:

dependence on anatomic location and function. J Biomech. 1987;20:105561.

35. Doadrio JC, Arcos D, Vallet-Reg M, et al. Calcium sulphate-based

cements containing cephalexin. Biomaterials. 2004;25:262935.

36. Phaechamud T, Charoenteeraboon J. Antibacterial activity and

drug release of chitosan sponge containing doxycycline hyclate.

AAPS Pharm Sci Tech. 2008;9:82935.

37. Zhang Y, Zhang M. Calcium phosphate/chitosan composite

scaffolds for controlled in vitro antibiotic drug release. J Biomed

Mater Res. 2002;62:37886.

38. Hunt CD. One possible role of dietary boron in higher animals

and humans. Biol Trace Elem Res. 1998;66:20525.


1 3


13/13

39. Ying X, Cheng S, Peng L, et al. Effect of boron on osteogenic

differentiation of human bone marrow stromal cells. Biol Trace

Elem Res. 2011;144:30615.

40. Devirian TA, Volpe SL. The physiological effects of dietary

boron. Crit Rev Food Sci Nutr. 2003;43(2):21931.

41. Brown RF, Rahaman MN, Huang WH, et al. Effect of to borate

glass composition on its conversion hydroxyapatite and on the

proliferation of MC3T3-E1 cells. J Biomed Mater Res Part A.

2008;88:392400.

42. Benderdour M, Bui-Van T, Dicko A, et al. In vivo and vitro

effects of boron and boronated compounds. J Trace Elem Med

Biol. 1998;12(1):27.

43. Murray FJ. A human health risk assessment of boron (boric acid

and borax) in drinking water, biochemical and physiologic con-

sequences of boron deprivation in humans. Regul Toxicol Phar-

macol. 1995;22:22130.

44. Forrest HN. Boron in human and animal nutrition. Plant Soil.

1997;193:199208.

45. Murray FJ. A comparative review of the pharmacokinetics of

boric acid in rodents and humans. Biol Trace Elem Res. 1998;

66:3319.


1 3

MaterialsInMedicine2013 Sandra Alcaraz

Documents

Transcript of MaterialsInMedicine2013 Sandra Alcaraz