Strontium doped injectable bone cement for potential

Materials Science and Engineering C 80 (2017) 93–101

Contents lists available at ScienceDirect

Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Strontium doped injectable bone cement for potential drug delivery applications Ali Taha a,b, Muhammad Akram c, Zaidoon Jawad b, Ammar Z. Alshemary d, Rafaqat Hussain e,⁎ a

Department of Chemistry, Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor, Darul Ta'zim, Malaysia Department of Chemistry, Faculty of Science, University of Misan, Misan, Iraq Department of Chemistry, Government Degree College, Raiwind (Lahore), Pakistan d Department of Biomedical Engineering, Faculty of Engineering, Karabuk University, 78050 Karabuk, Turkey e Department of Physics, COMSATS Institute of Information Technology, Islamabad, Pakistan b c

a r t i c l e

i n f o

Article history: Received 24 October 2016 Received in revised form 8 May 2017 Accepted 16 May 2017 Available online 17 May 2017 Keywords: Brushite Dicalcium phosphate Antibiotics Strontium Drug delivery

a b s t r a c t Microwave assisted wet precipitation method was used to synthesize calcium deficient strontium doped βtricalcium phosphate (Sr-βTCP) with a chemical formula of Ca2.96-xSrx(PO4)2. Sr-βTCP was reacted with monocalcium phosphate monohydrate [Ca(H2PO4)2.H2O, MCPM] in presence of water to furnish corresponding Sr containing brushite cement (Sr-Brc). The samples were characterized by using X-ray diffractometry (XRD), Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM). Strontium content in the prepared samples was determined by using inductively coupled plasma optical emission spectrometry (ICP-OES). The effect of Sr2+ ions on the structural, mechanical, setting properties and drug release of the cement is reported. Incorporation of Sr2+ ions improved the injectability, setting time and mechanical properties of the Brc. The release profiles of antibiotics incorporated in Brc and Sr-Brc confirmed that the Sr incorporation into the Brc results in the efficient release of the antibiotics from the cement. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Calcium phosphate-based cements such as dicalcium phosphate (DCP) has received considerable attention of researchers due to their potential applications in dental, maxillofacial and orthopaedic surgery [1,2]. In addition, DCP is able to harden in situ and present appropriate mechanical properties [3,4]. Adequate mechanical strength, good setting time and the ability to inject the cement into a defect are now considered characteristics of paramount importance for minimally invasive surgery [5,6]. The use of trace elements in a biomaterial is an interesting alternative to growth factors when one wants to favor bone formation. Strontium is known to improve bone formation in osteoporotic patients [7–10], which is naturally present in the mineral phase of bone (1 wt%), especially in regions of high metabolic turnover [11]. The partial replacement of Ca2+ ions by Sr2+ ions can improve the mechanical properties [12] and the dissolution of the material [13]. Strontium is considered as a bone-seeking element that presents a beneficial effect on bone growth [14]. Its ability to decrease bone resorption and to enhance bone formation in vivo has also been proven [15–17]. Synthetic methods such as solid state calcination, co-precipitation, sol gel process and hydrothermal method [18–20] have been extensively studied for the preparation of Sr-TCP. Sr2+ ions have the ⁎ Corresponding author. E-mail address: [email protected] (R. Hussain).

http://dx.doi.org/10.1016/j.msec.2017.05.117 0928-4931/© 2017 Elsevier B.V. All rights reserved.

same physiological and chemical behavior as Ca2+, which can be embedded into the mineral structure of bone by ion substitution in place of Ca2+ [21]. Recent studies on Zn- and ZnSr-substituted brushite cements injected into trabecular bone defects in pigs proved that Zn and Sr are good inductors of osteoprogenitor cell proliferation and differentiation [22]. Results have indicated that Sr is a more potent inhibitor of osteoclastic activity than Zn, as much fewer osteoclast-like cells could be found in ZnSr-containing implants. Several studies have also shown that Sr-containing cements are more osteoconductive than Zn-containing cements [11,23,24]. Recently, strontium ranelate, a newly developed drug treating osteoporosis, has shown to have dual effects of stimulating osteoblast differentiation and inhibiting osteoclast activity (bone resorption), which could reduce the incidence of fractures in osteoporotic patients [16]. In addition, the partial substitution of Ca2+ by Sr2+ can apparently improve the biological properties of calcium phosphate based materials [25]. In vitro studies of SOAS cells treated with 625 μg/mL of Sr-HA (Sr10(PO4)6OH2) showed no signs of apoptosis [26]. In addition, Sr doped hydroxyapatite (Ca5Sr5(PO4)6(OH)2)/chitosan composite scaffolds showed good osteoconductivity [27]. Osteomyelitis is a bone infection usually caused by bacteria, mycobacteria, or fungi. Its treatment mainly involves operative debridement, removal of all foreign bodies and antibiotic therapy [28,29]. The inability to maintain high antibiotic concentrations at the site of infection is the major cause of failure in the treatment of this disease. A

94

A. Taha et al. / Materials Science and Engineering C 80 (2017) 93–101

good approach to overcome this situation is to fill the defect with an antibiotic-loaded bone graft together with the further local administration of the drug whenever required. Different types of biomaterials have been used as local drug delivery systems in resistant cases of osteomyelitis [30]. Ceramic-based local drug delivery systems have been suggested as potential materials for the treatment of osteomyelitis. Most drug-delivery systems in clinical use are based on ceramic materials such as nanoporous alumina [31], silicon carbide [32] and calcium phosphates [33]. Many studies have investigated combinations of therapeutic agents with different calcium phosphates such as sintered hydroxyapatite (HA) [34], precipitated amorphous calcium phosphate [35] and calcium polyphosphates [36]. The success of this idea was favored by the easy incorporation of pharmaceutical and biological substances into the cement solid or liquid phases, the intimate adaptation of the cement paste to bone defects and the high cement porosity that permits the release of the entrapped substance to the local environment [37]. Furthermore, the low-temperature setting of CPCs allows the incorporation of heat-labile medicaments and substances into the cement matrix during its preparation. Water is part of the setting reaction of brushite cement and enables adjustment of the cement porosity, a determinant factor for the release kinetics of the loaded drug [38]. Investigation of osteotransductive brushite-based materials as carriers for antimicrobial agents [39,40] has been motivated by the difficulty of treating bone infections due to the poor accessibility of antibiotics to the infection site and the formation of antibiotic-resistant biofilms [41–44]. In this study, an array of dicalcium phosphate cement containing Sr2+ ions was prepared and the influence of Sr2+ doping on the setting time, injectability, compressive strength, porosity and drug release from dicalcium phosphate cement is reported. 2. Materials and methods All chemicals for the synthesis of βTCP were purchased form Qrec (New Zealand) and were of reagent grade, whereas MCPM (Ca(HPO4)2.H2O, 99.9%, MW 234.05) was purchased from Sigma-Aldrich (USA). Gentamicin sulfate, Ampicillin, and Amoxicillin trihydrate were sourced from Science Lab (Texas, USA). Phase purity, lattice parameters and degree of crystallinity were evaluated by using Bruker D8 Advance X-ray diffractometer (XRD), the diffractogram was recorded between 2θ range of 20° - 80° at room temperature with the step size of 0.02° and step time of 1 s. The crystallinity noted by Xc corresponds to the fraction of crystalline βTCP phase in the investigated volume of powdered sample by using Xc = 1- V300/0210 / I0210, where I0210 is the intensity of (0 2 10) reflection of βTCP structure and V300/0210 is the intensity of the hollow between the (3 0 0) and (0 2 10) reflections. Lattice parameters were calculated by using Unit Cell software (program UnitCell-method of TJB Holland & SAT Redfern 1995). Morphology and elemental composition were studied by FESEM (Zeiss-LEO 1530) attached with Energy Dispersive X-Ray Analysis (EDX) (Oxford instrument, Swift ED 3000) operated at 20 kV. Readings at 5 different locations were recorded to calculate the average elemental composition. Functional groups were identified by FTIR analysis carried out on FTIR Nicolet iS50 spectrometer by using classic KBr pellet technique. The spectra were recorded in a wavenumber range of 400–4000 cm−1 in transmission mode with 32 scans and resolution of 4 cm−1. 2.1. Preparation of Sr doped β-tricalcium phosphate βTCP and Sr-βTCP were synthesized by a microwave assisted wet precipitation method. Different samples were prepared with a Ca/P and (Ca + Sr)/P molar ratio of 1.48 (Table 1). In a typical reaction, calcium nitrate (Ca(NO3)2·4H2O) was dissolved in (100 mL) double distilled water. Diammonium hydrogen phosphate ((NH4)2HPO4) was added dropwise under constant stirring to the solution of calcium nitrate, pH of the solution was adjusted to 7 by using ammonium hydroxide and

Table 1 Molar quantities of reactants used for the synthesis of βTCP and Sr-βTCP. Sample ID

Ca(NO3)2.4H2O (mol)

(NH4)2HPO4 (mol)

Sr(NO3)2 (mol)

Sr (wt%)

β-TCP 1Sr-βTCP 2Sr-βTCP 3Sr-βTCP 4Sr-βTCP

8.88 8.63 8.38 8.13 7.88

6 6 6 6 6

0.00 0.25 0.50 0.75 1.00

– 2.4 6.6 7.4 8.9

the mixture was refluxed in a microwave oven (SHARP, model R218LS) at 800 W for 5 min. The resulting suspension was filtered, washed with double distilled water, dried at 80 °C for 17 h and calcined at 1000 °C for 2 h to produce βTCP. The synthesis of four different strontium substituted β-Ca3(PO4)2 compositions were prepared according to the procedure given above. In brief, calcium-deficient apatites containing Sr2+ ions were synthesized via a microwave assisted wet precipitation route by the slow addition of (NH4)2HPO4 solution to the continuously stirred (1000 rpm) solution mixture of Ca(NO3)2.4H2O and Sr(NO3)2. The wt% of Sr in the sample varied between 2.4 and 8.9 (Table 1). The pH of the mixed solution/suspension was maintained at 7.4 by adding the required amounts of 8 M ammonium hydroxide (NH4OH) solution. 2.2. Cement preparation βTCP/Sr-βTCP (1.00 g) and MCPM (1.00 g) were thoroughly mixed and the liquid phase, water (1 mL) was added to the powder. The resulting mixture was mixed until a homogenized paste was achieved (Eq. 1). Ca3 ðPO4 Þ2 þ Ca ðH2 PO4 Þ2 H2 O þ 7H2 O→4CaHPO4 2H2 O

ð1Þ

2.3. Setting time Initial and final setting times of the cements were measured by using Gillmore needle (ASTM C266–89). Powder phase components were mixed in a mortar for about 2 min and then mixed with liquid phase thoroughly to form a homogeneous paste. The cement paste was poured into a split Teflon mold of 6 mm diameter and 12 mm height. A needle of 2.12 mm diameter and 113.4 g was placed on the cement sample [6]. Initial setting time was recorded when the needle could not leave an impression on the surface of the cement paste. Similarly, a needle of 1.06 mm diameter and 453.6 g weight was used to determine the final setting time. The setting time reported is an average of 3 measurements. 2.4. Injectability of dicalcium phosphate cement The paste of DCP cements was introduced in a commercial syringe with an aperture of 2 mm in diameter (13 mm diameter cartridge with a nominal capacity of 10 mL). A 5 kg compressive load was then mounted vertically on the top of the plunger to start the injection. Injections were carried out until the paste was no longer injectable. The percentage injectability was calculated by applying Eq. 2[45]: Inj% ¼ ðW F − WA Þ=ðW F − WE Þ 100

ð2Þ

Where Inj% is the percentage injectability, WF is the weight of the syringe full of paste and WA is the weight of the syringe after the injection and WE is the weight of the empty syringe.


2.5. Compressive strength testing

95

Table. 2 Lattice parameters and degree of crystallinity of different βTCP and Sr-βTCP samples.

Cylindrical samples of DCP cement were cast in a Teflon mold (6 mm diameter and 12 mm height). The hardened samples were removed from the mold and were carefully polished using an 800 grit SiC sandpaper to smooth the ends to achieve a height of 12 mm according to ASTM F451-99a. The specimens were immersed in 50 mL of SBF solution for 1, 3, and 7 days [46]. The compressive strength of the specimen dried at room temperature for 24 h was measured by using INSTRON Series X1S Automated Materials Tester-Version 8.33.00 at a crosshead speed of 0.5 mm/min. The results of compressive strength are expressed as the mean of 5 values.

Samples

βTCP 1Sr-βTCP 2Sr-βTCP 3Sr-βTCP 4Sr-βTCP

Chemical formula

Ca8.88(PO4)6 Ca8.63Sr0.25(PO4)6 Ca8.38Sr0.5(PO4)6 Ca8.13Sr0.75(PO4)6 Ca7.88Sr1(PO4)6

Lattice parameter

Degree of crystallinity % XC

a-Axis (Å)

c-Axis (Å)

Cell Vol. (Å)3

10.343 10.367 10.378 10.390 10.398

37.393 37.453 37.671 37.832 37.972

3481.74 3499.54 3524.67 3539.36 3547.16

81.77 80.87 78.99 75.12 72.28

2.6. Density and porosity The apparent density was measured by using the Archimedes principle (Eq.3). The porosity of the material was calculated from the division of the apparent density by the skeletal density (Eq.4), skeletal density used in the calculations were 2.33 g/cm3 for brushite and 2.92 g/cm3 for monetite [47]. pa ¼

A ðp°−dÞ þ d A−B

ð3Þ

Na2SO4 reagents in deionized water in accordance with kokubo's specification [48]. The pH of the solution was adjusted at 7.25 by the addition of hydrochloric acid, HCl. The samples were kept in SBF at 37 °C for up to 7 days. The whole volume of the SBF was extracted for measurement of its Ca2+ and Sr2+ content and then fed with fresh solution again. The Ca2+ and Sr2+ concentrations were determined by using Flame Atomic Absorption Spectrometer (Perkin Elmer AAnalyst 400) [49]. 2.8. Determination of drug release profiles in vitro

pa = apparent density, A = weight in air, B = weight in water, p° = density of water 1 g/cm3, d = density of air (approx. 0.001 g/cm3). Φð%Þ ¼

p 1− a 100 ps

ð4Þ

Φ = porosity, pa = apparent density, ps = skeletal density.

The weight amount of βTCP, MCPM and antibiotic (1:1:0.05) were used as a powder phase to fabricate cement paste. The liquid (L) was added to the powder (P) phase and the resulting mixture was hand mixed for 2 min until a homogenized paste was achieved. Samples for the drug release study were prepared by placing the cements paste into cylindrical Teflon molds (6.0 mm diameter × 12 mm height). The drug loaded samples were immersed in 15 mL of phosphate buffered

2.7. In vitro ion release DCP cement samples for the evaluation of the ion release were prepared by placing the cement paste into cylindrical Teflon molds (6.0 mm diameter × 12 mm height). After the preparation, samples were allowed to set for 24 h at 25 °C. The set samples were immersed in SBF solution. The SBF solution had a chemical composition and concentration similar to the inorganic part of human plasma and was prepared by dissolving NaCl, KCl, K2 HP4, 3H2O, MgCl2.6H2O, CaCl2, and

Fig. 1. XRD pattern of βTCP and Sr doped βTCP.

Fig. 2. FTIR spectrum of βTCP and Sr doped βTCP.

96


saline (PBS) and incubated at 37 °C. 2 mL aliquots of the solution were taken directly from the vessels after 30 min, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 72, 168 and 336 h. The aliquots were analyzed by recording

UV absorbance of the samples at 332 nm, 334 nm and 230 nm to detect the presence of gentamicin sulfate, amoxicillin, and ampicillin trihydrate respectively.

Fig. 3. FESEM and EDX images showing the morphology of (a) βTCP, (b)1Sr-βTCP, (c)2SrMg-βTCP, (d)3SrMg-βTCP and (e) 4Sr-βTCP calcination at 1000 °C.


3. Results and discussion

Table 3 Lattice parameters of Sr-substituted brushite and monetite cements.

3.1. Phase analysis of βTCP and Sr-βTCP Synthesis of βTCP through a microwave assisted wet precipitation method involved the preparation of calcium deficient apatitic structure [Ca8.88(HPO4)(PO4)5(OH)] by exposing the reaction mixture with Ca/P molar ratio of 1.48 to 800 W microwave radiations for 5 min. Calcination of this calcium deficient structure at 1000 °C for 2 h furnished βTCP, SrβTCP samples were prepared in exactly the same manner except the initial Ca + Sr/P ratio was adjusted to 1.48 to ensure successful incorporation of the Sr2+ ions into the structure (Eqs 5 and 6). 8:88‐XCaðNO3 Þ2 þ XSrðNO3 Þ2 þ 6ðNH4 Þ2 HPO4 þ6NH4 OH→ ðCa8:88‐x Srx Þ ðHPO4 ÞðPO4 Þ5 ðOHÞþ18NHNO3 þ5H2 O

ð5Þ Ca8:88‐x Srx ðHPO4 ÞðPO4 Þ5 ðOHÞ

97

1000 °C=2h

→

3ðCa1‐x Srx Þ2:96 ðPO4 Þ2 þH2 O

ð6Þ

Where X = [0.00–1.00] βTCP is known to crystallize into the rhombohedral space group R3c, where the unit cell contains 21 calcium ions and 14 PO4 groups (Ca/P = 1.5) [50]. In a unit cell of βTCP there are five crystallographically independent sites, Ca(1)–Ca(5) [51]. Site M (5) is located on a 3 fold axis with no shared PO4 edges and is the preferred site of occupancy by the dopant requiring octahedral geometry [50]. Fig. 1 shows the X-ray diffraction pattern of samples after annealing to 1000 °C. All diffraction peaks of the synthesized βTCP were corresponding to the βTCP phase (JCPDS 09–0169). The incorporation of Sr into the βTCP lattice resulted in a shifting of diffraction peaks to lower 2θ values and reduction in the intensity of the XRD peaks, which was ascribed to the substitution of smaller sized Ca2+ ions (0.99 A°) with larger sized Sr2+ ions (1.12 A°) (Fig. 1). The highest peak in βTCP phase is formed at 2θ = 31.392° which corresponds to (0 2 1 0) plane. In the XRD pattern of 1Sr-βTCP, 2Sr-βTCP, 3Sr-βTCP and 4Sr-βTCP the corresponding peak of (0 2 10) plane appeared at 2θ = 31.341, 31.290, 31.239 and 31.188 respectively [52]. Degree of crystallinity of the synthesized Sr-βTCP samples (Table.2) decreased with increase in Sr2+ substitution, which can be attributed to increased lattice strain caused by larger Sr2+ ion replacing the smaller Ca2+ ion in the βTCP lattice [53]. Lattice parameters a and c increased with the addition of Sr to βTCP due to the higher ionic radii of Sr2+ in comparison with Ca2+ (Table 2) [53,54].

Fig. 4. XRD patterns of brushite and Sr substituted brushite and monetite cements.

Samples ID

Lattice parameters a(A°)

b(A°)

c(A°)

Cell Volume (A°)3

Brc 1Sr-Brc 2Sr-Brc 3Sr-Moc 4Sr-Moc

5.808 5.802 5.806 6.854 6.521

15.126 15.158 15.138 6.850 6.868

6.234 6.442 6.236 7.058 7.481

490.465 491.292 490.978 319.818 312.510

FTIR spectra of Sr2+ doped βTCP calcined at 1000 °C are shown in (Fig. 2). The bands at 534 and 623 cm−1 were attributed to the bending mode (ʋ4) of the O–P–O bonds. The bands associated with the stretching modes (ʋ3 and ʋ1) of P\\O bonds were observed at 934 cm−1, 1030 and 1124 cm−1. The bands at 1640 and 3445 cm−1 were assigned to the adsorbed water. Incorporation of Sr2+ ion in βTCP samples resulted in the broadening and shifting of phosphate band from 969 cm−1, 1124 cm−1 to 941 cm−1 and 1152 cm−1 due to the reduction in the degree of crystallinity [55]. Absence of peaks at 630 cm−1 and 3570 cm−1 confirmed that our samples were free of hydroxyapatite as a secondary phase. FESEM micrographs of pure βTCP powders calcined at 1000 °C showed that the particles existed as tightly packed irregular shaped agglomerates (Fig. 3a), however, the morphology of βTCP was changed to loosely packed clusters upon Sr2+ doping (Fig. 3-e). EDX data confirmed the presence of Sr2+ in the samples, the observed concentration of the dopant was in close agreement with the theoretical value, hence confirming the suitability of the microwave assisted wet precipitation method to prepare Sr2+ doped βTCP. XRD data along with FTIR and EDX analysis confirmed that the Sr2+ substitution had taken place inside the crystal structure rather than the mere adsorption of Sr2+ ion on the surface of βTCP (Fig. 3). 3.2. Phase analysis of Brushite and Sr-Brushite/Monetite cement The X-ray diffraction patterns of the Brc, 1Sr-Brc and 2Sr-Brc (Fig. 4) showed that the cement matrix was predominantly composed of crystalline brushite (JCPDS 72–0713). XRD patterns further confirmed that the Sr2+ doping resulted in the formation of monoclinic crystalline

Fig. 5. Infrared spectra of Brc, Sr doped brushite and monetite cements.

98


Fig. 6. FESEM images showing the morphology of (a) Brc, (b)1Sr-Brc, (c) 2Sr-Brc, (d) 3Sr-Moc and (e) 4 Mg-Moc.

Fig. 7. Setting time and injectability of strontium substituted DCP cements (values are mean ± SD; n = 5).


99

Table 4 Release of Ca2+ and Sr2+ ions in SBF over 7 days at 37 °C. Immersion time (days)

1 3 7

Release of Ca2+ ion (mg/L) and Sr2+ ion (mg/L) in SBF Brc

1Sr-Brc

Ca2+

Ca2+

Sr2+

2Sr-Brc Ca2+

Sr2+

Ca2+

Sr2+

Ca2+

Sr2+

4.658 4.624 4.662

11.035 13.275 11.921

0.18 0.48 0.22

13.207 15.035 13.650

3.799 5.620 4.575

10.264 15.854 11.757

3.502 8.991 4.614

10.330 13.227 10.665

3.477 5.181 2.665

phase with reduced lattice parameters (Table 3). A minor peak observed at 31.08° in the XRD pattern was assigned to the (0 2 1 0) plane of the unreacted βTCP. Shifting of peaks to lower diffraction angles was also observed in the XRD pattern, which was ascribed to the substitution of smaller sized Ca2+ ions with larger sized Sr2+ ions in the DCP structure. The increase in the Sr2+ ion concentration in the sample resulted in the formation of monetite. The XRD pattern of 3Sr-Moc and 4Sr-Moc contained peaks at 26.50°, 28.75°, 30.22°, 32.48°, 36.15°, 40.35° and 49.31°, which were respectively assigned to the (200), (− 1 − 12), (− 120), (201), (0 − 22), (003) and (− 320) planes of crystalline monetite (JCPDS 71–1760). A higher Sr2+ content resulted in the formation of monetite as a main setting product, which could be attributed to the change of lower pH value in the cement pastes [56]. Previous studies have shown that the excess of acidity in the cement paste, as well as a lower rate of calcium phosphate precipitation, favors the formation of monetite in a cement matrix [11]. FTIR of Sr-Brc and Sr-Moc are shown in (Fig. 5). FTIR spectra of the Sr doped cements contained bands at 3540 cm−1, 3485 cm−1, 3290 cm−1 and 3155 cm−1, which were assigned to the O\\H stretching of water. PO stretching was observed at 1139 cm−1, 1057 cm−1, and 988 cm−1. The P–O(H) stretching was found at 864 cm−1, PO bending was recorded at 664 cm−1, 575 cm−1 and 520 cm−1. The intensity of vibrational bands decreased upon incorporation of Sr2+ ions considerably suggesting a distortion of the structure, altering the specific geometric linkage of the Ca\\P related bonds due to the presence of Sr2+. The FESEM images of Sr doped DCP cements are presented in Fig. 6. FESEM of pure brushite showed the formation of small structured particles of irregular morphology (Fig. 6a), which changed to the loosely packed plate like morphology with heterogeneous size distribution upon Sr2+ doping (Fig. 6b–e). The plates did not show a specific orientation, suggesting a more or less isotropic behavior.

3Sr-Moc

4Sr-Moc

4. In vitro study 4.1. Setting time and injectability The initial and final setting times of Sr-DCP at room temperature are presented in Fig. 7. Initial and final setting time for 1Sr-Brc and 2Sr-BrC were 12, 16 min and 21, 25 min respectively, However, increasing the concentration of Sr resulted in the formation of monetite phase (3SrMoC and 4Sr-MoC) with considerably reduced setting times (Fig. 7). Results indicate that the Sr content in DCP can influence the setting reaction, giving rise to the formation of different cement compositions [57]. The effect of Sr on the injectability of the DCP is illustrated in (Fig. 7). Injectability of the paste increased from 10% to 66% (1Sr-Brc) and then to 69% (2Sr-Brc) upon increasing the Sr content, however, a further increase in the Sr content resulted in the formation of monetite with reduced injectability (Fig. 7). 4.2. Ion release from Sr-substituted brushite/monetite cements Calcium and strontium ions release in SBF over 7 days reported in Table 4 was monitored by ICP-OES. The results showed that the dissolution of samples initiated in the early stages of immersion in the SBF solution. Increase in the amount of Ca2+ and Sr2+ ion release in the SBF solution was observed from day 1 to day 3. However, a gradual decrease in Ca2+ and Sr2+ was observed after 7 days, which was ascribed to their probable consumption in the formation of apatite layer. This fluctuated trend in release profile of Sr2+ ion can have a beneficial influence on the proliferation and differentiation of the stem cells [58]. Effective release of Sr2+ ions from calcium phosphate cement can be exploited to treat bone defects provided that the in-vivo Sr2+ concentration is kept below the toxic limit. Our results have shown that the

Fig. 8. Compressive strengths of Brc, 1Sr-Brc, 2Sr-Brc, 3Sr-Moc and 4Sr-Moc before and after immersion in SBF for up to 7 days (values are mean ± SD; n = 5).

100


Fig. 9. In vitro release profiles of gentamicin sulfate, amoxicillin and ampicillin trihydrate from (a) Brc and (b) 2Sr-Brc.

release of Sr2+ ion reaches 8.99 mg/L for 3Sr-Moc after 3 days. This level is considered to be relatively non-toxic and it is well within the doses stated to positively affect osteoblast-like cells in vitro (0.1–5 mM) [59–62]. A study has shown that the treatment with 65 mg/L of Sr2+ (from SrCO3) for 12 days improved in vitro proliferation and differentiation of human mesenchymal stem cells [58]. A study has also demonstrated that the continuous treatment with 3 mM (≈26 mg/L) of Sr2+ (from strontium ranelate) for 21 days increases osteoblastic differentiation of Murine bone marrow stromal cells both in the presence and absence of dexamethasone [63]. Furthermore, Human primary osteoblasts cells when exposed to 5 mM (≈44 mg/L) or greater of Sr2+ (from strontium ranelate) for 21 days promote osteoblast maturation and an osteocyte-like phenotype [58,64]. 4.3. Compressive strength and porosity Fig. 8 shows the effect of immersion of samples in SBF on the compressive strength of the cement. In general, the compressive strength of the pure brushite cement increased with the increase in the immersion time, which was ascribed to the reduction in the porosity of the sample from 19.84% to 12.93% after 7 days of immersion. Incorporation of Sr into the sample resulted in the increase in the compressive strength from 1.32 MPa to 34 MPa (2Sr-Brc). In general, an increase in the compressive strength was observed for all samples after 7 days of immersion, which was ascribed to the reduction in the relative porosity of the samples [57]. The compressive strength of 3Sr-Moc and 4Sr-Moc was lower than those of 1Sr-Brc and 2Sr-Brc. Our injectibility tests and compressive strength data have shown that the Sr-DCP can be used to repair minimally invasive surgery for the repair of non-load bearing bone defects/injuries [65]. 4.4. In vitro drug release profiles Elution of antibiotic from bone cements can proceed by one or combinations of three processes: surface elution, diffusion and diffusion via newly formed cracks [66]. We have monitored the elution of drug from

Table 5 Relative porosity % of Brc, Sr-Brc and Sr-Moc immersion in SBF. Samples ID

Brc 1Sr-Brc 2Sr-Brc 3Sr-Moc 4Sr-Moc

Relative porosity% 0d

1d

3d

7d

28.62 27.152 15.98 20.92 24.83

19.84 21.10 20.25 27.12 20.95

18.58 25.08 29.73 30.21 27.88

12.93 18.41 19.18 20.47 22.01

the scaffolds for 14 days to study the short and long term release profiles of drugs, Initially the release was followed closely every hour to assess the rapid release of the physically adsorbed drug from the surface of the samples. This was followed by the monitoring of samples after 1, 3, 4, 7 and 14 days to determine the release of residual drug from the pores of the samples. The burst release in the initial phase followed by a slow release over 14 days is considered favorable to prevent bacterial infection after the surgery. The cumulative in vitro drug release of 65%, 57% 47% from Brc was respectively observed for gentamicin sulfate, amoxicillin and ampicillin trihydrate (Fig. 9a). Increase in drug release to 96%, 87% and 73% was recorded in the first 72 h for gentamicin sulfate, amoxicillin and ampicillin trihydrate respectively upon Sr doping (Fig.9b). Low degree of porosity observed in Sr-Brc cement when compared with Brc was attributed to a greater release of gentamicin sulfate (Table 5). 5. Conclusions We have successfully employed microwave assisted wet precipitation method to dope βTCP with Sr2+ ions, which was used to prepare strontium doped brushite and monetite cement. The final setting time of Brc prepared at 2 g/mL was 4 min, which was increased to 21 min upon Sr doping. Slower setting time resulted in an increase of injectability to 66%. Sr2+ ions improved the drug release profiles when compared to pure Brc, indicating that the cement can be an effective drugs carrier under in vitro conditions. The in vitro release of antibiotics was bimodal, an initial burst release was observed followed by a diffusion mediated sustained release. Drug release profiles of the antibiotics loaded onto the Sr doped Brc showed that the SrBrc cements can be effective drugs carriers under the in vitro conditions. We have successfully demonstrated the formation of a hand-mixed injectable inorganic matrix capable of sustained release of therapeutic compounds that also has adequate mechanical properties. References [1] S. Raynaud, E. Champion, D. Bernache-Assollant, P. Thomas, Biomaterials 23 (2002) 1065–1072. [2] J. Zhang, W. Liu, V. Schnitzler, F. Tancret, J.-M. Bouler, Acta Biomater. 10 (2014) 1035–1049. [3] F. Tamimi, Z. Sheikh, J. Barralet, Acta Biomater. 8 (2012) 474–487. [4] K. Jamuna-Thevi, F.A. Zakaria, R. Othman, S. Muhamad, Mater. Sci. Eng. C 29 (2009) 1732–1740. [5] E. Fernández, F.J. Gil, S.M. Best, M.P. Ginebra, F.C.M. Driessens, J.A. Planell, J. Biomed. Mater. Res. 41 (1998) 560–567. [6] M. Roy, K. DeVoe, A. Bandyopadhyay, S. Bose, Mater. Sci. Eng. C 32 (2012) 2145–2152. [7] E. Boanini, M. Gazzano, A. Bigi, Acta Biomater. 6 (2010) 1882–1894. [8] J.E. Barralet, L.M. Grover, U. Gbureck, Biomaterials 25 (2004) 2197–2203. [9] K. Kawabata, T. Yamamoto, A. Kitada, Phys. B Condens. Matter 406 (2011) 890–894.

A. Taha et al. / Materials Science and Engineering C 80 (2017) 93–101 [10] S.R. Kim, J.H. Lee, Y.T. Kim, D.H. Riu, S.J. Jung, Y.J. Lee, S.C. Chung, Y.H. Kim, Biomaterials 24 (2003) 1389–1398. [11] M. Hamdan Alkhraisat, C. Moseke, L. Blanco, J.E. Barralet, E. Lopez-Carbacos, U. Gbureck, Biomaterials 29 (2008) 4691–4697. [12] M. Huang, T. Li, N. Zhao, Y. Yao, H. Yang, C. Du, Y. Wang, Mater. Chem. Phys. 147 (2014) 540–544. [13] G. Romieu, X. Garric, S. Munier, M. Vert, P. Boudeville, Acta Biomater. 6 (2010) 3208–3215. [14] S. Pors Nielsen, Bone 35 (2004) 583–588. [15] W. Querido, A.L. Rossi, M. Farina, Micron 80 (2016) 122–134. [16] M. Schumacher, A. Lode, A. Helth, M. Gelinsky, Acta Biomater. 9 (2013) 9547–9557. [17] N. Neves, B.B. Campos, I.F. Almeida, P.C. Costa, A.T. Cabral, M.A. Barbosa, C.C. Ribeiro, Mater. Sci. Eng. C 59 (2016) 818–827. [18] H.B. Pan, Z.Y. Li, W.M. Lam, J.C. Wong, B.W. Darvell, K.D.K. Luk, W.W. Lu, Acta Biomater. 5 (2009) 1678–1685. [19] H. Zhou, S. Kong, Y. Pan, Z. Zhang, L. Deng, Mater. Sci. Eng. C 56 (2015) 174–180. [20] O. Kaygili, S. Keser, Mater. Lett. 141 (2015) 161–164. [21] X.B. Chen, D.R. Nisbet, R.W. Li, P.N. Smith, T.B. Abbott, M.A. Easton, D.H. Zhang, N. Birbilis, Acta Biomater. 10 (2014) 1463–1474. [22] F. Tamimi, B. Kumarasami, C. Doillon, U. Gbureck, D.L. Nihouannen, E.L. Cabarcos, J.E. Barralet, Acta Biomater. 4 (2008) 1315–1321. [23] K.K. Johal, G. Mendoza-Suarez, J.I. Escalante-Garcia, R.G. Hill, I.M. Brook, J. Mater. Sci. Mater. Med. 13 (2002) 375–379. [24] M.H. Alkhraisat, C. Rueda, J. Cabrejos-Azama, J. Lucas-Aparicio, F.T. Marino, J. Torres Garcia-Denche, L.B. Jerez, U. Gbureck, E.L. Cabarcos, Acta Biomater. 6 (2010) 1522–1528. [25] Z. Gu, B. Huang, Y. Li, M. Tian, L. Li, X. Yu, Mater. Sci. Eng. C 61 (2016) 526–533. [26] Y. Lei, Z. Xu, Q. Ke, W. Yin, Y. Chen, C. Zhang, Y. Guo, Mater. Sci. Eng. C 71 (2017) 653–662. [27] M. Frasnelli, F. Cristofaro, V.M. Sglavo, S. Diré, E. Callone, R. Ceccato, G. Bruni, A.I. Cornaglia, L. Visai, Mater. Sci. Eng. C 72 (2017) 134–142. [28] C.F. Marques, A. Lemos, S.I. Vieira, OAB da Cruz e Silva, A. Bettencourt, J.M.F. Ferreira, Ceram. Int. 42 (2016) 2706–2716. [29] Ž. Radovanović, B. Jokić, D. Veljović, S. Dimitrijević, V. Kojić, R. Petrović, D. Janaćković, Appl. Surf. Sci. 307 (2014) 513–519. [30] D. Pastorino, C. Canal, M.-P. Ginebra, Acta Biomater. 12 (2015) 250–259. [31] M. Karlsson, E. Pålsgård, P.R. Wilshaw, L. Di Silvio, Biomaterials 24 (2003) 3039–3046. [32] L. Treccani, T. Yvonne Klein, F. Meder, K. Pardun, K. Rezwan, Acta Biomater. 9 (2013) 7115–7150. [33] A.N. Shuid, N.I. Ibrahim, M.C.I.M. Amin, I.N. Mohamed, Curr. Drug Targets 14 (2013) 1558–1564. [34] A. Ślósarczyk, J. Szymura-Oleksiak, B. Mycek, Biomaterials 21 (2000) 1215–1221. [35] A. Dion, B. Berno, G. Hall, M.J. Filiaggi, Biomaterials 26 (2005) 4486–4494. [36] A. Dion, M. Langman, G. Hall, M. Filiaggi, Biomaterials 26 (2005) 7276–7285. [37] D. Campoccia, L. Montanaro, P. Speziale, C.R. Arciola, Biomaterials 31 (2010) 6363–6377. [38] M.P. Hofmann, A.R. Mohammed, Y. Perrie, U. Gbureck, J.E. Barralet, Acta Biomater. 5 (2009) 43–49. [39] U. Gbureck, E. Vorndran, J.E. Barralet, Acta Biomater. 4 (2008) 1480–1486.

101

[40] U. Gbureck, E. Vorndran, F.A. Müller, J.E. Barralet, J. Control. Release 122 (2007) 173–180. [41] G. Laverty, S.P. Gorman, B.F. Gilmore, 2 - Biofilms and Implant-Associated Infections, in: L. Barnes, I.R. Cooper (Eds.), Biomaterials and Medical Device - Associated Infections, Woodhead Publishing, Oxford 2015, pp. 19–45. [42] B.F. Gilmore, L. Carson, 8 - Bioactive biomaterials for controlling biofilms, in: L. Barnes, I.R. Cooper (Eds.), Biomaterials and Medical Device - Associated Infections, Woodhead Publishing, Oxford 2015, pp. 163–183. [43] S. Fujimura, T. Sato, T. Kikuchi, J. Zaini, K. Gomi, A. Watanabe, J. Orthop. Sci. 14 (2009) 658–661. [44] H. Zazo, C.I. Colino, J.M. Lanao, J. Control. Release 224 (2016) 86–102. [45] E.B. Montufar, Y. Maazouz, M.P. Ginebra, Acta Biomater. 9 (2013) 6188–6198. [46] A. Forouzandeh, S. Hesaraki, A. Zamanian, Ceram. Int. 40 (2014) 1081–1091. [47] J. Engstrand Unosson, C. Persson, H. Engqvist, J. Biomed. Mater. Res. B Appl. Biomater. 103 (2015) 62–71. [48] T. Kokubo, H. Takadama, Biomaterials 27 (2006) 2907–2915. [49] S. Hesaraki, S. Farhangdoust, K. Ahmadi, R. Nemati, M. Khorami, J. Aust. Ceram. Soc. 48 (2012) 166–172. [50] L.W. Schroeder, B. Dickens, W.E. Brown, J. Solid State Chem. 22 (1977) 253–262. [51] M. Yashima, A. Sakai, T. Kamiyama, A. Hoshikawa, J. Solid State Chem. 175 (2003) 272–277. [52] S. Kannan, F. Goetz-Neunhoeffer, J. Neubauer, S. Pina, P.M.C. Torres, J.M.F. Ferreira, Acta Biomater. 6 (2010) 571–576. [53] M. Kavitha, R. Subramanian, R. Narayanan, V. Udhayabanu, Powder Technol. 253 (2014) 129–137. [54] O. Kaygili, S. Keser, M. Kom, Y. Eroksuz, S.V. Dorozhkin, T. Ates, I.H. Ozercan, C. Tatar, F. Yakuphanoglu, Mater. Sci. Eng. C 55 (2015) 538–546. [55] L. He, G. Dong, C. Deng, Ceram. Int. 42 (2016) 11918–11923. [56] Z. Sheikh, Y.L. Zhang, L. Grover, G.E. Merle, F. Tamimi, J. Barralet, Acta Biomater. 26 (2015) 338–346. [57] D. Guo, K. Xu, X. Zhao, Y. Han, Biomaterials 26 (2005) 4073–4083. [58] S.R.K. Meka, S. Jain, K. Chatterjee, Colloids Surf. B: Biointerfaces 146 (2016) 649–656. [59] A. Barbara, P. Delannoy, B.G. Denis, P.J. Marie, Metabolism 53 (2004) 532–537. [60] J. Braux, F. Velard, C. Guillaume, S. Bouthors, E. Jallot, J.-M. Nedelec, D. LaurentMaquin, P. Laquerrière, Acta Biomater. 7 (2011) 2593–2603. [61] S.C. Verberckmoes, M.E. De Broe, P.C. D'Haese, Kidney Int. 64 (2003) 534–543. [62] M. Schumacher, A. Henss, M. Rohnke, M. Gelinsky, Acta Biomater. 9 (2013) 7536–7544. [63] S. Choudhary, P. Halbout, C. Alander, L. Raisz, C. Pilbeam, J. Bone Miner. Res. Off. J. Am. Soc. Bone Miner. Res. 22 (2007) 1002–1010. [64] G.J. Atkins, K.J. Welldon, P. Halbout, D.M. Findlay, Osteoporos. Int.: a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA 20 (2009) 653–664. [65] M.P. Ginebra, E.B. Montufar, 10 - Injectable biomedical foams for bone regeneration, in: P.A. Netti (Ed.), Biomedical Foams for Tissue Engineering Applications, Woodhead Publishing 2014, pp. 281–312. [66] W.C. Liu, C.T. Wong, M.K. Fong, W.S. Cheung, R.Y. Kao, K.D. Luk, W.W. Lu, J. Biomed. Mater. Res. B Appl. Biomater. 95 (2010) 397–406.