cephalexin-loaded injectable macroporous calcium phosphate bone cement

Cephalexin-Loaded Injectable Macroporous CalciumPhosphate Bone Cement

Saeed Hesaraki, Roghayeh Nemati

Ceramic Department, Materials and Energy Research Center, Tehran, Iran

Received 16 January 2008; revised 13 April 2008; accepted 14 July 2008Published online 29 September 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31222

Abstract: Different types of calcium phosphate cements (CPCs) have been studied as

potential matrices for incorporating different types of antibiotics. All of these matrices were

morphologically microporous whereas macroporosity is essential for rapid cement resorption

and bone replacement. In this study, liberation of cephalexin monohydrate (CMH) from a

macroporous CPC was investigated over 0.5–300 h in simulated body fluid and some

mathematical models were fitted to the release profiles. Macroporosity was introduced into the

cement matrix by using sodium dodecyl sulfate molecules as air-entraining agents and the

effect of both surfactant and CMH on basic properties of the CPC was studied. Incorporation

of CMH into the CPC composition increased the setting time, decreased the crystallinity of the

formed apatite phase, and improved the injectability of the paste. The use of both CMH and

sodium dodecyl sulfate did not affect the rate of conversion of the reactants into apatite phase

while soaking the cements in simulated body fluid. Results showed that the liberation rate of

the drug from porous CPC was higher than that of the nonporous CPC but same release

patterns were experienced in both types of cements, that is, like to nonporous CPC, a time-

dependent controlled release of the incorporated drug was obtained from macroporous CPC.

The Weibull model was the best fitting-equation for release profiles of all cements. The

liberated CMH was as active as fresh cephalexin. It is concluded that this macroporous CPC

can be successfully used as drug carrier with controlled release profile for the treatment of

bone infections. ' 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 89B: 342–352,

2009

Keywords: calcium phosphate(s); drug release; bone graft; hydroxyapatite

INTRODUCTION

The use of prosthetic devices for treatment of bone inju-

ries/illnesses is continuously expanding with an increas-

ingly active and aging population. The synthetic bone

grafts have been extensively used to treat bone and dental

defects.1–4 However, the healing rate and therefore the suc-

cesses of these materials are largely dependent on preven-

tion of further bacterial growth in defect site. Thus, the

localized delivery of antibiotics from a suitable matrix was

developed and its efficacy in improving the treatment pro-

cedure was approved.5 The calcium-containing cements

such as calcium sulfate and calcium phosphate cements

(CPCs) are potential materials for these purposes.6,7 CPCs

are biocompatible and osteoconductive materials widely

used in bone defect treatments.8–10 Because of their indi-

vidual properties such as formability, self-setting ability invivo, injectability, precipitation of bone-like apatite phase

in physiologic temperature, and isothermal setting reac-

tions, CPCs are potential matrices for incorporating differ-

ent drugs. The release behavior of different types of

antibiotics such as gentamicim, vancomycin, tobramycin,

tetracycline, and doxycycline from calcium phosphate

cement (CPC) matrices has been studied previously.11–18

Cephalexin, a semisynthetic cephalosporin antibiotic, is

also bactericidal compound and has been shown to be

active against most gram-positive strains of microorgan-

isms. It is effective in wide variety of infections including

skin, bone,19–21 joints,22 tooth,23 and other organs. Some

authors reported the release behavior of cephalexin or other

cephalosporins from different cement-type matrices includ-

ing polymethyl metacrylate,18 resin-based bioglass

cements,24 and calcium sulfate-based cements.7,25 However,

there are a few articles deal with the use of hydroxyapatite

cement as chephalosporin (especially cephalexin) carrier.

Yu26 and Otsoka27 loaded 0.9–4.8 wt % of cephalexin and

norfoloxacin as drug model into a hydroxyapatite cement

and discussed the antibiotic release behavior by Higuchi’s

model. In addition, the CPCs matrices used and character-

ized in all studies were structurally microporous while

macroporosity is essential for accelerating the cement

Correspondence to: S. Hesaraki (e-mail: [email protected])

' 2008 Wiley Periodicals, Inc.

342

resorption and bone replacement rate.28–30 In other words,

the macroporosity allows penetration of blood vessels as

well as migration and proliferation of osteoblasts, osteo-

clasts and other cells into the bulk of the implant resulted

in matrix deposition in the empty spaces.31–33

In this study, injectable and macroporous calcium phos-

phate cement was prepared using surfactant additive and

used as drug carrier. The release behavior of the cephalexin

from this matrix was truly described by different mathe-

matical models. The novel aspect of this study deals (i)

with the influence of the macroporosity (the pores with di-

ameter large enough for cell invasion) on the extent of

cephalexin release and on the transport properties of the

matrix and (ii) with the influence of the loaded antibiotic

on the physico-chemical characteristics of this sodium do-

decyl sulfate (SDS)-containing matrix.

MATERIALS AND METHODS

Preparation of CPCs

Tetracalcium phosphate (TTCP) was synthesized as

described elsewhere34 briefly, by solid-state reaction

between 1 mole of calcium carbonate and 1 mole of dical-

cium phosphate anhydrate (both from Merck, Germany) at

15008C for 6 h. The product was ground in a planetary

mill until a fine powder was obtained. The solid phase of

the cement was a calcium phosphate powder consisting of

TTCP and dicalcium phosphate dihydrate (DCPD), at molar

ratio of 1 to 1. Solution of 6 wt % Na2HPO4 (in distilled

water) was selected as liquid phase of the cement. The

CPC paste was made by mixing the powder phase with the

liquid phase at a powder to liquid (P/L) ratio of 3.8 g/mL.

Macroporosity was introduced into the cement matrix by

the method described by Sarda et al.35 Briefly, for prepar-

ing macroporous CPCs, aqueous solution of 6 wt %

Na2HPO4 and 20 or 100 mM SDS surfactant was used as

liquid phase of the cement. For preparing antibiotic-con-

taining CPCs, cephalexin monohydrate (CMH), a semisyn-

thetic cephalosporin antibacterial agent with a chemical

structure like to that of shown in Figure 1, was homoge-

nized with the solid phase of the cement. Noted that the

weight of the added cephalexin was ignored during calculat-

ing the P/L ratio. The samples were named based on the

amount of incorporated CMH and SDS as shown in Table I.

Particle Size of the Reactants

The particle size of the DCPD and the ground TTCP was

determined in isopropanol medium using a laser particle size

analyzer instrument (Fritsch particle sizer analysette 22).

Surface Charge in Aqueous Solutions

The Zeta potential (n) values of the reactants in contact

with different aqueous solutions were measured by the

method described previously.36 In brief, TTCP and DCPD

reactants were separately dispersed in various aqueous solu-

tions, including Na2HPO4, Na2HPO4/SDS, and Na2HPO4/

CMH at a solid loading of 0.005 wt %. The stabilized sus-

pension was analyzed by Zeta-Sizer 3000HS via laser

Doppler velocimetry. The measurement was repeated four

times for each sample.

Physical/Physico-Chemical Properties of the CPCs

The influence of SDS and CMH on the physical/physico-

chemical properties of the CPCs was determined by charac-

terizing initial setting time, compressive strength (CS),

injectability, density, porosity, and specific surface area of

the cements.

The initial setting time (Ist) of the CPCs was recorded in

a humid chamber (EHRET, KBK 4200, Germany) at 378Cand 100% humidity using a Gillmore needle test with a

needle weight of 113.5 g and tip diameter of 2.1 mm

according to ASTM C266-99-A standard. The paste was

considered to set when the needle did not form a visible

print onto the specimen. Five specimens of each composi-

tion were tested.

For CS measurements, the powder phase was mixed

with the liquid phase to form a paste. Then, the paste was

poured into a Teflon mold, and compacted with a 10 N

force to form a cylindrical sample with a diameter of

6 mm and a height of 12 mm. After setting, the specimen

was removed from the mold, part of the samples stored in

an incubator at 378C and 100% relative humidity and part

Figure 1. Chemical structure of cephalexin monohydrate (CMH).

TABLE I. Naming CPCs According to Concentration of SDSin the Liquid Phase and Amount of Drug Incorporated Into theSolid (Powder) Phase

Amount of Drug Incorporated

Into the CPCs (wt %)

[SDS] (mM) 0 1 5 10

0 S0-D0 S0-D1 S0-D5 S0-D10

20 S20-D0 S20-D1 S20-D5 S20-D10

100 S100-D0 S100-D1 S100-D5 S100-D10

343CEPHALEXIN-LOADED INJECTABLE MACROPOROUS BONE CEMENT

Journal of Biomedical Materials Research Part B: Applied Biomaterials

of samples were immersed in 25 mL of a simulated body

fluid (SBF) with ionic concentration (142.0 mM Na1,

5.0 mM K1, 1.5 mM Mg21, 2.5 mM Ca21, 147.8 mMCl2, 4.2 mM HCO3

2, 1.0 mM HPO422, and 0.5 mM SO4

22)

resembling that of human blood plasma.37 The SBF was

prepared by dissolving reagent-grade NaCl, KCl,

NaHCO3, MgCl2.6H2O, K2HPO4.3H2O, Na2SO4, and

CaCl2 in distilled water and buffered at pH 7.4 by tris-

(hydroxymethyl aminomethane) and HCl. The CS of wet

specimen (five specimens of each composition) was meas-

ured at a crosshead speed of 1 mm/min after 24 h of incu-

bation at 378C and 1 day immersion in SBF (378C) usinga static mechanical testing device (Zwick/Roell-HCR 25/

400, Germany).

To evaluate the injectability (I) of the CPCs, a paste of

each composition was made by adding the powder phase to

the liquid phase and the obtained paste was mixed for 15 s.

Eight grams of the paste was transferred into a 10 mL sy-

ringe (internal tip diameter of 800 lm) and was extruded

by a compressive load mounted vertically on top of the

plunger using a computerized universal testing machine

(Zwick/Roell-HCR 25/400) at a crosshead speed of 15 mm/

min and a maximum load of 100 N. The mass of the paste

extruded through the syringe was recorded and the inject-

ability was calculated using the following expression38:

Ið%Þ ¼ ðMass of the cement paste extruded through syringeÞðThe initial mass of the pasteÞ

3 100 ð1Þ

Four specimens of each composition were tested.

To estimate the bulk density and micro/macro/total po-

rosity of the cements, the specimens were prepared in the

same way as the CS test specimens. Five specimens were

prepared for each cement composition. The bulk density

(db) was obtained using Eq. (2):

db ¼ MCPC

VCPC

¼ MCPC

pr2CPCHCPC

ð2Þ

where MCPC is the mass of the specimen, VCPC its volume,

rCPC its radius and HCPC its height. Total porosity (Ptotal)

measurements were performed on incubated dried speci-

mens according to the density method.38 Ptotal was calcu-

lated using the expression:

ptotal ¼ 100 1� dbdp

� �ð3Þ

where dp is the powder density of the CPC obtained by

pycnometry technique. For pycnometry measurements, the

CPC was ground and passed through an 800 sieve. Macro-

porosity (Pma) was calculated using the following expression:

pma ¼ 100 1� dbd0

� �ð4Þ

where d0 is the bulk density of the dried specimen without

SDS. Microporosity (Pmi) was obtained by the following

expression:

Pmi ¼ Ptotal � Pma ð5Þ

The specific surface area (S.S.A) of the cylindrical sam-

ples was measured by nitrogen adsorption according to the

Brunauer, Emmel and Teller method using Micromeritics,

Gemini 2375.

X-Ray Diffractometry and Crystallinity

The phase composition of the specimens were determined

by an X-ray diffractometer (Philips PW 3710 with Cu-Ka

radiation) after storing in an incubator for 24 h (378C and

100% humidity) or soaking in SBF for 1 and 7 days

(378C). After each mentioned period, the specimen was

washed with double distilled water, dried, ground to fine

powder and weighed to evaluate and compare phase transi-

tions occurred during the incubating/soaking periods.

Data were acquired from 10 to 508 2y with a scan rate of

0.02, 2y/s.To obtain the effect of both added SDS and CMH on

the crystallinity of the formed apatite, the peak of apatite

phase in plane (002) (2y 5 25.98) was recorded separately

a minimum of five times (0.05 2y/min). This analysis was

done for 1-day soaked specimens. The angular width of the

diffraction peak was measured at 1/2 of the height of the

maximum intensity above the background. The B value,

half width, was corrected for instrumental broadening by

Warren’s method using following equation39–41:

B2 ¼ b2 þ b2 ð6Þ

where b was the instrumental broadening and b was the

corrected peak width. Because, peak width inversely corre-

lates with crystal size and lattice perfection (i.e., the

smaller the width, the larger and/or less strained the crys-

tal) the 1/b value was used to relate the X-ray diffractome-

try (XRD) data more directly to these crystal parameters.

Scanning Electron Microscopy

The microstructure of the specimens was evaluated by

scanning electron microscopy (SEM). For this purpose, the

specimen was prepared in the same way as the CS one and

its fractured surfaces were coated with a thin layer of gold.

A scanning electron microscope (TESCAN, VEGA II,

XMU) with an accelerating voltage of 15 kV was

employed for this analysis.

Drug Release and Kinetic Study

The antibiotic release tests were done over 300 h. Five

dried specimens of each composition (disc shape, 3 mm in

344 HESARAKI AND NEMATI


height and 5 mm in diameter) were weighed and then sepa-

rately immersed in a closed dark-glassy bottles containing

25 mL of SBF (pH 5 7.4). The bottles were placed in an

orbital shaker and rotated at 40 rpm at 378C. At defined

time intervals (0.5, 1, 4, 7, 10, 24, 72, 168, and 300 h), the

whole volume of the SBF was extracted to identify the

released cephalexin and the bottles were fed with fresh

SBF. The drug released from the cement matrix into SBF

was determined by U.V. visible spectroscopy (Jenway

6500, UK) through measuring the absorbance number at

261 nm. The calibration curve was made by different

known diluted concentrations of cephalexin solutions.

To find out the mechanism of drug release, data

obtained from the in vitro release tests were fitted to various

kinetic equations, including Higuchi’s, Korsmeyer-Peppas’s

and Weibull’s. In these equations Ft, the cephalexin released

at time t, is expressed as a function of time.

Antibacterial Activity

To determine the activity of the liberated cephalexin, anti-

bacterial tests were done according to the standardized

method42 using strains of S. aureus (ATCC 29213) and

E. coli (ATCC 25922). For preparation of bacterial culture,

Mueller-Hinton agar plates were streaked with the bacterial

strain followed by incubation at 378C for 24 h. Bacterial

spores were added to 8 mL of Mueller-Hinton broth until a

suspension containing 2 3 108 CFU/mL was achieved.

From a 10 mg/mL original stock solution of fresh cepha-

lexin powder, serial stock dilutions was prepared and

mixed with the same volume of the broth in which the final

concentration of cephalexin in Mueller-Hinton broth

reached to the range of 0.015–32 lg/mL. Similarly, dilution

series were made by the release medium of the cephalexin-

containing S100-D10 matrix (after 0.5 h of elution). The

samples were incubated at 378C for 18 h. After the incuba-

tion, minimal inhibitory concentration (MIC) value was

expressed as the lowest concentration of the cephalexin

that inhibited bacterial growth judged by lack of turbidity

of samples.

Calculations and Statistical Analysis

Data were processed using Microsoft Excel 2003 software

and the results were produced as mean 6 standard devia-

tion of at least four experiments. Significance between the

mean values was calculated using standard software pro-

gram (SPSS GmbH, Munich, Germany) and the p � 0.05

was considered as significant.

RESULTS

Particle Size of the Reactants

The mean particle size values (d50) of TTCP and DCPD

were 10.1 and 5.2 lm, respectively. For DCPD, 70% of the

particles had a size lower than 12 lm, whereas this statisti-

cal value was 19 lm for TTCP.

Surface Charge in Aqueous Solutions

Table II represents the n values of TTCP and DCPD, main

components of the CPCs, in different aqueous mediums.

For both TTCP and DCPD powders, negative n values

were recorded in all solution mediums. The surface charges

of the reactants significantly increased with the presence of

SDS and CMH molecules in the solution.

Physical/Physico-Chemical Properties of the CPCs

The results of the measured setting times, CS and inject-

ability of the CPCs were presented in Table III. The setting

time was not influenced by adding the SDS molecules to

the cement composition and the CPC pastes with or with-

out SDS molecules was initially set at about 7 min. The

setting time was significantly increased by incorporating

TABLE II. Zeta Potential Values (mV) of TTCP and DCPDReactants in Various Solutions (pH 5 7.4)

Solution of

Na2HPO4

Solution of

Na2HPO4 and

SDS (20 mM)

Solution of

Na2HPO4 and

CMH (5.5 mM)a

TTCP 212.5 6 1.8b 220.1 6 2.5 222.3 6 2.4

DCPD 214.3 6 2.1 224.3 6 3.0 229.4 6 1.6

a Equivalent to maximum solubility of the used CMH.b Mean 6 S.D. (n 5 4).

TABLE III. Initial Setting Time, Compressive Strength and Injectability of CPCs

Ist (min)

CS of Incubated

CPCs (MPa)

CS of Soaked

CPCs (MPa) I (%)

S0-D0 7.1 6 0.5 9.3 6 1.2 16 6 1.7 63.5 6 2.8

S20-D0 8.1 6 0.2 4.5 6 0.4 9.2 6 0.8 96.8 6 2.1

S100-D0 7.6 6 0.4 3.1 6 0.3 7.3 6 0.6 97.9 6 2.0

S0-D5 12.3 6 0.7 10.1 6 1.0 12.8 6 1.1 76.4 6 3.5

S20-D5 12.6 6 1.1 5.7 6 0.5 7.5 6 0.5 90.3 6 3.1

S100-D5 13.2 6 0.8 3.4 6 0.3 5.9 6 0.4 92.3 6 3.3

S0-D10 16.8 6 1.2 11.0 6 0.9 13.2 6 0.7 -

S20-D10 17.6 6 1.3 6.3 6 0.7 8.0 6 0.5 -

S100-D10 16.3 6 1.6 4.2 6 0.4 5.1 6 0.8 -



CMH into the CPC composition but the values are accepta-

ble for clinical operations.

As shown in Table III, After 24 h of incubation, those

CPCs containing 5 and 10% cephalexin exhibited a higher

CS than those of drug-free ones. Compared to incubated

specimens, the CS values of all specimens significantly

improved by the soaking operation. The values of the drug-

containing CPCs were significantly lower than that of drug-

free ones due to their drug liberation and poor crystallinity.

The CS values of nonporous CPCs were significantly higher

than that of porous (SDS-containing) CPCs (p � 0.005).

In SDS-containing cements, whole of the paste was

nearly extruded through the syringe and no filter pressing

phenomenon was observed during injection process. The

results showed that the injectability was also improved by

adding CMH. The extrusion process of the cephalexin-con-

taining porous CPCs (CPCs with SDS) was more difficult

than that of cephalexin-free ones, due to their higher P/Lratio (note that the weight of CMH was not entered when

calculating the P/L ratio. In addition, CMH is poorly solu-

ble in water (2 g/L) and the incorporated antibiotic can not

dissolve completely in the liquid part of the cement). Com-

pared to the additive-containing CPCs, the S0-D0 showed

poor injection behavior and a considerable content of the

paste was not extruded through the syringe tube.

Table IV provides some information from density, po-

rosity, and S.S.A of the porous/nonporous CPCs with or

without cephalexin. The bulk density of the CPCs signifi-

cantly decreased by adding the surfactant molecules to the

cement composition and was further decreased when more

SDS was used. An adverse result was obtained for total

porosities. The calculated bulk density of the cephalexin-

containing CPCs was slightly higher than that of drug-free

ones, due to their higher P/L ratios during making the

pastes. The total volume of the micropores was signifi-

cantly decreased with increasing the macroporosity in the

cement structure. In addition, the multipoint S.S.A value of

the CPC matrix significantly increased when SDS mole-

cules was introduced into the cement composition.

XRD and Crystallinity

Figures 2 and 3 show the XRD diagrams of the CPCs with

and without SDS/CMH at different times of setting/soak-

ing. The patterns of the cement powder and the added drug

are shown for comparison too. The patterns of all incubated

CPCs (with or without additive) were similar to that of

cement powder and the CMH could not be detected. In all

samples, considerable amounts of the reactants (TTCP and

DCPD) were converted into apatite phase after 1 day of

soaking and apatite was the most predominant phase of the

cements after 7 days of soaking. It means that the presence

of both SDS and CMH molecules did not affect the rate of

conversion of the reactants into apatite product.

Figure 4 shows the crystallinity indexes of some CPCs

studied in this work. Because, the peak width inversely corre-

lates with crystallinity (i.e., the smaller the width, the larger

and/or less strained the crystal) the 1/b value was used to

relate the XRD data more directly to crystallinity. The crys-

tallinity was significantly decreased by adding SDS and fur-

ther reduction was achieved by incorporating CMH.

SEM

Figure 5 shows the SEM micrographs of the set S0-D5, S20-

D5, and S100-D5 specimens at low magnification. Pores with

large dimensions ([10 lm) were observed in both S20-D5

and S100-D5 specimens but larger pores were seen for S100-

D5. The same pictures were observed in other porous CPCs

(Figures are not shown). No macropores were observed in

the microstructure of CPCs without any surfactant [Figure

5(a)]. Here, the term ‘‘macroporosity’’ has been used as that

defined in biomaterials science, that is large enough for cell

invasion. SEM micrographs of the 1-day soaked specimens

were presented in Figure 6. An entanglement of well-grown

apatite crystals was observed in S0-D0 specimen [Figure

6(a)], whereas the size of the crystals in S20-D0 and S100-

D0 [Figure 6(b,c)] were much smaller than that of S0-D0. A

network of nanoapatite crystals containing micro/nano poros-

ity were seen in the microstructure of S0-D5 specimen

[Figure 6(d)]. However, for both S20-D5 and S100-D5 speci-

mens [Figure 6(e,f)], the crystals were too small and they

were not seen at this magnification and a cloud-like micro-

structure of these very small crystals were only seen.

Cephalexin Release and Kinetic Study

Figure 7(a) shows the cumulative release of cephalexin as

a function of time for the studied samples. About 33 wt %

TABLE IV. Density, Porosity and Specific Surface Area Values of CPCs With or Without CMH and SDS

Sample db (g/cm3) Ptotal (V%) Pma (V%) Pmi (V%) S.S.A (m2/g)

S0-D0 1.74 6 0.06a 43.4 6 1.1 0 43.4 6 0.9 –

S20-D0 1.42 6 0.03 52.6 6 0.3 18.3 6 1.6 34.3 6 0.5 –

S100-D0 1.22 6 0.08 59.3 6 2.3 29.8 6 4.4 29.5 6 1.4 –

S0-D5 1.81 6 0.04 39.6 6 1.2 0 39.6 6 1.2 –

S20-D5 1.50 6 0.05 49.4 6 2.5 17.1 6 2.4 32.3 6 1.77 –

S100-D5 1.29 6 0.06 56.5 6 4.0 28.7 6 3.2 27.8 6 1.4 –

S0-D10 1.86 6 0.04 35.8 6 2.3 0 35.8 6 2.3 11.8 6 2.6

S20-D10 1.55 6 0.06 46.5 6 1.5 16.6 6 1.3 29.9 6 1.4 23.3 6 3.4

S100-D10 1.33 6 0.03 54.1 6 2.1 28.4 6 2.4 25.4 6 1.1 25.1 6 2.1

a Mean values 6 S.D. (n 5 5).



of the incorporated cephalexin was released from S0-D10

(nonporous) specimen after this time, which was signifi-

cantly lower than that released from both S20-D10 and

S100-D10 (porous) specimens (40 and 47%, respectively).

At the end of the elution period (after 300 h), nearly 85–89

wt % of the incorporated cephalexin was released from

both S100-D10 and S20-D10 porous samples, which were

significantly (p � 0.05) higher than that of nonporous S0-

D10 sample (70 wt %). The percentage of the released

cephalexin from S20-D5 specimen was nearly the same as

that of other porous cements [Figure 7(a)]. Figure 7(b)

shows the rate of cephalexin release during the first 10 h of

elution. The release rate from all specimens was hardly

reduced during the first 1 h. No significant difference was

observed in release rates of S20-D10 and S100-D10 sam-

ples, whereas the release rate from S0-D10 specimen was

significantly lower than that of porous samples. The least

release rate belonged to the S20-D5 specimen because of

lower content of the antibiotic.

Figure 2. XRD patterns of (a) S0-D0 and (b) S0-D5 specimens afterdifferent times of setting/soaking.

Figure 3. XRD patterns of (a) S100-D0 and (b) S100-D5 specimensafter different times of setting/soaking.



Table V presents the fit parameters for the release of

cephalexin from porous/nonporous CPCs using different

mathematical models. Release of the antibiotic from all

matrices followed Korsmeyer-Peppas and Weibull’s equa-

tion much better than Higuchi’s one. Both release exponent

(n) and Weibull constant (d) in nonporous cement were

nearly the same as those of porous cements, whereas the

rate constant values (K and s) were significantly different.

Antibacterial Activity

Table VI shows the MICs values corresponding to the fresh

cephalexin and liberated cephalexin for E. coli and S. aur-eus strains. No significant difference was found between

the MIC values of the fresh cephalexin and the liberated

one. For both compounds, the gram-positive strain S. aur-eus was more sensitive than E. coli.

DISCUSSION

An important limitation of many localized drug delivery

systems is a rapid release of the drug occurring within the

early hours of implantation. To maintain an effective thera-

peutic range, controlled release of drug should be adminis-

trated; otherwise an ineffective therapy is achieved by

rapid absorption of the released drug results in initial high-

peak plasma level. CPCs are potential substrates for load-

ing different types of antibiotics because of time-dependent

controlled release of loaded drug from these matrixes.

This study evaluated the release of cephalexin antibiotic

from a highly porous calcium phosphate cement matrix in

which the porosity was induced into the cement structure

using surfactant molecules.

Cephalexin was chosen for release experiments in the

present study, as it is a great gram positive coverage with

some activity against negative bacteria. It is effective in

wide variety of infections, including bone, joint, skin, and

teeth because of its high therapeutic/toxic ratio.

To obtain a macroporous paste during injection and set-

ting processes, SDS, a biocompatible anionic surfactant,

which widely used in cardiovascular applications and phar-

maceutical preparations,35,43–45 was chosen as additive. De-

velopment of macroporosity during setting would allow

fast bone ingrowth and appropriate osteointegration of the

cement.35

The mechanism of pore formation by using surfactant

molecules has been described previously.35 In brief, the air

bubbles entered into the cement paste during mixing the

powder with the liquid are covered by a sheath of surfac-

tant molecules and stabilized because of ‘‘strong affinity of

the anionic heads of the SDS molecules for calcium

ions.’’35

Figure 4. The crystallinity index (1/b) of some CPCs with or without

CMH.

Figure 5. SEM pictures of incubated CPCs with or without SDS molecules: (a) S0-D5, (b) S20-D5, and(c) S100-D5 (scale bar 5 500 lm).



Several experiments were performed to clear the influ-

ence of both surfactant and the loaded antibiotic on the ba-

sic characteristics of the cement matrix.

The results cleared that the growth of apatite crystals

was influenced by adding SDS molecules to the cement

composition. Further reduction was achieved by using

CMH. This result was in accordance with that of Sarda’s

et al.35 They experienced small crystallites of apatite phase

when using surfactant molecules in a tricalcium phosphate-

based cement. Similarly, poorly crystalline apatite phase

was obtained in SDS-containing CPCs of this study. The

setting reaction of the TTCP/DCPD-based cements is a dis-

solution-controlled process during the early stage of reac-

tion. In this stage, dissolution of the reactant in the liquid

phase leads to formation of a medium around the particles

that is supersaturated with respect to hydroxyapatite. This

leads to precipitation of apatite phase over the surfaces of

the reactants. At the second stage, further growth of apatite

crystals occurs through a diffusion-controlled mechanism,

which improves the mechanical strength of the CPC. The

presence of SDS molecules did not affect the degree of

supersaturation around the reactant particles, because the

same initial setting times were achieved for both SDS-con-

taining and SDS-free CPCs. However, adsorption of these

molecules onto the surfaces of the apatite nuclei inhibited

their further growth. Thus, the number of created apatite

crystals is increased by addition of the surfactant to the

CPC whereas their size is smaller that of surfactant-free

CPC.

The results showed that the use of CMH decreased the

crystallinity of the apatite too. Cephalexin is a carboxylic

acid type molecule (Figure 1). There are several studies

address the ability of carboxylic acid molecules (such as

citric acid) to adsorb onto the surfaces of reactant and/or

apatite nuclei of CPCs and to inhibit/retard further growth

of the precipitated phase.46 Thus, the increased setting time

of the cephalexin-containing CPCs suggest that is related to

adsorption of cephalexin molecules onto the surfaces of the

products hydroxyapatite (HA), which retards the heteroge-

neous nucleation process of apatite phase at the second

stage of conversion of the reactants into HA, where further

growth of the precipitated phase occurs through a diffu-

sion-controlled mechanism. Nonetheless, no inhibition was

observed in conversion of the reactants into apatite phase

during soaking the cephalexin-containing cements. It means

that more small-sized crystals have been formed.

Injectability was another characteristic of the CPC

changed by adding both SDS and CMH. Injectability is an

Figure 6. SEM pictures of 1-day soaked CPCs: (a) S0-D0, (b) S20-D0, (c) S100-D0, (d) S0-D5,

(e) S20-D5, and (f) S100-D5 (scale bar 5 2 lm).



important requirement of CPCs when using them as bone

filler in defected sites of difficult accessibility. It can be

improved by retarding the setting process or by modifying

the reactant particle interactions. It is also decreased with

increasing the solid content of the paste. A chemical mech-

anism can be assumed to act for improving the injectability

of cephalexin/surfactant-containing CPC pastes. These mol-

ecules can dissolve in reaction medium and adsorb onto the

surfaces of the calcium phosphate particles, increase parti-

cle surface charges and modify the interaction between

them by producing an electrostatic repulsion. This sugges-

tion was confirmed by increasing the surface charges of the

reactant particles in both SDS and CMH solution mediums

(Table II).

Since in all CPC systems studied as drug carrier the rate

of the cement resorption (degradation) was much lower

than the rate of drug release, the authors47 have described

that drug release from a CPC matrix is a diffusion-con-

trolled process.

The use of Higuchi’s equation yields a good regression

(R2 5 0.985) for the first 10 h of the release time. How-

ever, the practical data showed important deviation from

this equation when the entire dilution time was considered

(Table V). Although, better regression was achieved by

using the Korsmeyer-Peppas model, this model is suitable

to describe accurately the release process at short times48

(as very good regression (R2 5 0.992) is achieved by using

this model for the first 24 h of the release). In contrast,

when considering whole duration time, drug release fol-

lowed, in all cases, an exponential relationship (Weibull

equation) to the elution time as indicated by a higher corre-

lation coefficient compared to the Korsmeyer-Peppas model

(Table V). In Korsmeyer-Peppas model (power law), ‘‘K’’is an experimentally determined parameter depends on the

structural and geometrical characteristic of the dosage ma-

trix and ‘‘n’’, the release exponent, depends on the drug

release mechanism. Weibull function is the most appropri-

ate one to describe the drug release process at the entire

duration. In this model, ‘‘s’’ and ‘‘d’’ are real numbers

related to the specific surface area and mass transport char-

acteristics of the matrix, respectively.

Nearly, the same values of release exponent (n)/Weibull

constant (d) were achieved for the models fitted to experi-

mental data of all cement matrices. However, both K and svalues were significantly different. The value of s is related

to specific surface area and can be a function of the drug

particles moved inside the matrix pores. These confirmed

by the experimental data, that is the presence of macro-

pores and consequently the higher surface area values (Ta-

ble IV), the lower s value and easier mass transport in the

matrix pores. Thus, it can be expressed that the differences

in the release kinetic of the matrices was related to the dif-

ferences in their structural properties, like macroporosity,

surface area, pore size, and tortuosity. All of these are dif-

fusion-controlling factors.

Figure 7. (a) Cumulative release of cephalexin from porous or non-porous CPCs and (b) the rate of cephalexin released from the

porous or nonporous CPC matrices during the first 10 h of elution.

TABLE V. Fit Parameters for the Liberated Cephalexin From Different CPCs Using Various Models

Higuchi (Ft 5 Kt0.5) Korsmeyer-Peppas (Ft 5 K tn) Weibull (Ft 5 100 (12exp(2(t/s)d)

K R2 K N R2 s D R2

S0-D10 4.97 (0.12)a 0.601 14.29 (0.49) 0.27 (0.01) 0.977 157.38 (6.59) 0.37 (0.03) 0.997

S20-D10 6.10 (0.21) 0.593 18.45 (1.15) 0.28 (0.01) 0.972 66.99 (2.98) 0.39 (0.02) 0.998

S100-D10 6.41 (0.19) 0.416 21.34 (0.82) 0.30 (0.02) 0.963 44.13 (4.19) 0.40 (0.01) 0.989

S20-D5 5.67 (0.26) 0.543 17.72 (0.32) 0.28 (0.01) 0.976 88.50 (3.12) 0.37 (0.02) 0.998

a The results are expressed as mean 6 (S.D) (n 5 5).



Another important point, which should be considered in

this work, is the low values of n (�0.3) and d (0.37–0.39)

of the related models. Although estimates for n � 0.5 and

d � 0.75 indicate Fickian diffusion in disc-shape CPC mat-

rices,49 the low values of n and d indicates that drug

release is suggested to affect by some physico-chemical

phenomena interfering with drug transport slowing down

the release. These phenomena are: (i) formation of cepha-

lexin-calcium chelate complexes through interaction of the

carboxyl group of the cephalexin molecules with the cal-

cium ions of the matrix, (ii) Precipitation of apatite phase

onto the reactants and drug particles during soaking (elu-

tion process) the samples in release medium results in

changing the cement morphology, and (iii) Poor water solu-

bility of the cephalexin in room temperatures (2 g/L may

be dissolved readily, but higher concentrations are obtained

with increasing difficulty). The last effect is more tangible

after the first 24 h of the elution. Note that drug solubility

is an important factor that affects the release kinetic. In

other words, the drug loaded into the matrix should be

firstly dissolved and then transport through the liquid

phase. It can be suggested that dissolution of the drug in

the pores was another controlling factor delaying the

release process.

Biological activity of the liberated drug is an important

criterion for capability of a drug matrix. There are several

studies that approve the compatibility of the antibiotics

with calcium phosphate matrices.50,51 As observed in this

study, CMH was also compatible with these matrices and

maintained its ability to inhibit bacterial growth. The activ-

ity of the antibiotic was not affected even by the presence

of SDS molecules.

CONCLUSIONS

In this study, the release behavior of CMH from a macro-

porous CPC was determined. Effect of added drug on the

basic properties of the cement was also cleared. The rate of

drug liberation from a CPC matrix was significantly influ-

enced by the presence of macroporosity without any change

in the release mechanism. Thus, macroporosity did not

limit the controlled release of the cephalexin and a long-

term drug release could be achieved in both porous and

nonporous cement. CMH was compatible with the CPC

matrix and maintained its ability to inhibit bacterial growth

even at the presence of SDS molecules. Both SDS surfac-

tant and cephalexin antibiotic affect the basic properties of

the CPC such as mechanical strength, crystallinity of the

formed apatite phase, porosity and morphology except to

setting time, which was only influenced by added cepha-

lexin. This macroporous CPC can be successfully used as

antibiotic carrier for the treatment of osteomyelitis.

The authors acknowledge Mrs. F. Ghaderi, Mr. Hafezi-

Ardakani, Mr. Zamanian, and Mr. Jabbari for their help in

this work.

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