Materials Science and Engineering C 49 (2015) 106–113

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Calcium phosphate bone cements for local vancomycin delivery Dagnija Loca ⁎, Marina Sokolova, Janis Locs, Anastasija Smirnova, Zilgma Irbe Rudolfs Cimdins Riga Biomaterials Innovations and Development Centre of Riga Technical University, Pulka 3, LV-1007 Riga, Latvia

a r t i c l e

i n f o

Article history: Received 11 August 2014 Received in revised form 2 December 2014 Accepted 22 December 2014 Available online 24 December 2014 Keywords: Calcium phosphate cement PLA Vancomycin Microspheres Drug delivery

a b s t r a c t Among calcium phosphate biomaterials, calcium phosphate bone cements (CPCs) have attracted increased attention because of their ability of self-setting in vivo and injectability, opening the new opportunities for minimally invasive surgical procedures. However, any surgical procedure carries potential inflammation and bone infection risks, which could be prevented combining CPC with anti-inflammatory drugs, thus overcoming the disadvantages of systemic antibiotic therapy and controlling the initial burst and total release of active ingredient. Within the current study α-tricalcium phosphate based CPCs were prepared and it was found that decreasing the solid to liquid phase ratio from 1.89 g/ml to 1.23 g/ml, initial burst release of vancomycin within the first 24 h increased from 40.0 ± 2.1% up to 57.8 ± 1.2% and intrinsic properties of CPC were changed. CPC modification with vancomycin loaded poly(lactic acid) (PLA) microcapsules decreased the initial burst release of drug down to 7.7 ± 0.6%, while only 30.4 ± 1.3% of drug was transferred into the dissolution medium within 43 days, compared to pure vancomycin loaded CPC, where 100% drug release was observed already after 12 days. During the current research a new approach was found in order to increase the drug bioavailability. Modification of CPC with novel PLA/vancomycin microcapsules loaded and coated with nanosized hydroxyapatite resulted in 85.3 ± 3.1% of vancomycin release within 43 days. © 2014 Elsevier B.V. All rights reserved.

1. Introduction In the last decades musculoskeletal diseases and disorders are becoming a great problem all over the world and every year the number of patients suffering from these diseases dramatically increases [1,2]. Although the development of medications and techniques directed to the treatment of these diseases, even in the initial stages, is in great progress, still there are some unclear issues regarding the balance between maximal drug efficiency and minimal side effects [3,4]. The most prospective materials for the bone tissue replacement and regeneration are calcium phosphates (CaP) due to their biocompatibility, osteoconductivity and similarity to the natural bone mineral phase [5,6]. Among CaP biomaterials, CaP bone cements have attracted extended attention because of their ability of self-setting in vivo, moldability and injectability, opening the new opportunities for minimally invasive surgical procedures [2,7,8]. However, any surgical procedure carries potential inflammation and bone infection risks, traditionally prevented through prolonged (2–6 weeks) systemic antibiotic therapy [9–11]. The main disadvantages of systemic therapy are that only a small fraction of any given dose actually reaches the surgical site, thus the use of local or site specific antibiotic delivery system could be a solution to achieve high

⁎ Corresponding author. E-mail addresses: [email protected] (D. Loca), [email protected] (M. Sokolova), [email protected] (J. Locs), [email protected] (A. Smirnova), [email protected] (Z. Irbe).

http://dx.doi.org/10.1016/j.msec.2014.12.075 0928-4931/© 2014 Elsevier B.V. All rights reserved.

drug levels at an infection site and to minimize systemic side effects [12,13]. If compared to the drug delivery systems based on bioceramic materials, where drugs can be added using different strategies, like sorption, physical/mechanical aspects or chemical linking, drugs in the CPC can be easily dispersed throughout the entire cement matrix [4, 14]. However, it should be considered that CPC setting reaction, porosity and mechanical properties can be considerably affected by the antibiotic introduction within the calcium phosphate bone cement [15,16]. Three basic strategies can be used for the drug incorporation into calcium phosphate bone cements, like mixing the drug with cement solid phase or dissolving the active substance in the liquid phase, impregnation of CPC with drug solution or modifying the CPC with microencapsulated drug forms. The main advantages of the third strategy over the others, is based on the possibility to obtain long term drug delivery systems, at the same time decreasing the initial burst release, characteristic for the calcium phosphate bone cements [17]. In spite of all the advantages, calcium phosphate bone cements possess poor degradability, limiting the bone regeneration rate and low mechanical properties, limiting their use in load-bearing applications [18–20]. To overcome the limitations of poor degradability, CPCs can be modified with polymers, which upon the degradation could release the acidic monomers, enhancing CPC degradation [21–23]. Among the various polymer systems, poly(lactic acid) (PLA) and its copolymers with glycolic acid have been used as macroporosity inducers (in the form of microspheres, microcapsules or microfibers) [24–26], not only because of their degradation product acidic nature,

D. Loca et al. / Materials Science and Engineering C 49 (2015) 106–113

but also due to their biocompatibility, tailorable properties and approval from the Food and Drug Administration to be used in clinics [27,28]. In order to overcome the disadvantages of systemic therapy, the poor degradability of the CPC and to ensure prolonged (2–6 weeks) antibiotic delivery to the targeted site, in the current research two strategies of vancomycin incorporation into the CPC matrix were compared. Local drug delivery systems were prepared either dispersing the drug throughout the CPC matrix, estimating the solid to liquid phase ratio changes onto the drug release kinetics and cement properties, or modifying the CPC with microencapsulated vancomycin forms, evaluating the effect of nanosized hydroxyapatite addition during the microencapsulation process onto the drug release profile and cement properties. 2. Materials and methods 2.1. Materials Poly(lactic acid) (PLA) (Biomer L9000) with molecular weight of 200–300 kDa and polyvinyl alcohol (PVA) with molecular weight of 25 kDa (98 mol% hydrolyzed) were purchased from Polysciences (Warrington, FL). Vancomycin hydrochloride (from streptomyces orientalis), dichloromethane (≥99.8%), orthophosphoric acid (≥85%), calcium oxide (≥97%) and isopropyl alcohol (≥99.7) were purchased from Sigma-Aldrich (St. Louis, MO). Sodium dihydrogen phosphate and sodium hydrogen phosphate were purchased from Acros Organics (Geel, Belgium). 2.2. Synthesis of nanosized hydroxyapatite (HAp) Calcium phosphate powders where synthesized using wet precipitation reaction between calcium hydroxide and orthophosphoric acid as described previously [29]. The concentrations of Ca(OH)2 suspension and H3PO4 solution used for the synthesis process were 0.45 M and 2.00 M respectively. The obtained precipitates were vacuum filtered and the final concentration of HAp suspension was 0.2 g/ml.

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2.5. Characterization of microcapsules Microanalysis (apparatus — Vario MACRO CHNS, Hanau, Germany) was used to determine the nitrogen content in samples and the total drug load (DL) in microparticles was calculated according to Eq. (1): DLð%Þ ¼

Nel  100; Ntot

ð1Þ

where Nel is the nitrogen content found using microanalysis and Ntot is the calculated nitrogen content in vancomycin hydrochloride. The average microcapsule size and particle size distribution were determined using a laser particle size analyzer (ANALYSETTE 22, measuring range from 0.01–1000 μm, laser wavelength 650 nm). Each sample was measured in triplicate. The surface morphology and inner structure of microcapsules were examined using scanning electron microscopy (SEM, Tescan Mira \LMU, Czech Republic) at an acceleration voltage of 3–7 kV. Each sample was sputter coated with gold prior to imaging. 2.6. Preparation of calcium phosphate bone cements CPC solid phase was α-tricalcium phosphate powder (α-TCP), prepared by heating a mixture of CaCO3 and CaHPO4 (molar ratio 1:2) at 1300 °C for 4 h with subsequent quenching in air. The obtained αTCP was milled in a planetary ball mill (Fritsch, Pulverisette 5, Germany) for 1 h at 320 rpm in isopropyl alcohol. Cement liquid phase was a mixture of 0.5 M Na2HPO4 and 0.5 M NaH2PO4 solutions (volume ratio 20:1). The CPC samples with solid to liquid phase ratios of 1.89 g/ml, 1.75 g/ml and 1.23 g/ml were used in this study. In order to prepare calcium phosphate bone cement composites, all solid additives were properly mixed prior to the addition of liquid phase. The solid and liquid phases of the CPC were intensively mixed for 30 s and then placed into the teflon molds (7 mm in diameter and 16 mm in height). For CPC compositions and their detailed preparation see Table 1.

2.3. Preparation of vancomycin hydrochloride loaded PLA microcapsules

2.7. Characterization of calcium phosphate bone cements

Vancomycin hydrochloride loaded PLA microcapsules were prepared using the slightly modified double emulsification technique described previously [30]. Briefly, PLA (1 g) was dissolved in 10 ml of dichloromethane. PVA (4 g) was dissolved in 100 ml of water. Vancomycin hydrochloride (1 g) was added to the polymer solution in dichloromethane. S/O (solid phase/organic phase) primary suspension was properly homogenized for 30 s at 7000 rpm and added to 100 ml of 4% aqueous PVA solution. S/O/W (solid phase/organic phase/water phase) double emulsion was homogenized for 30 s at 7000 rpm. After emulsification, the organic solvent was extracted in 2 l of water for 30 min. Then the microcapsules formed were separated by centrifugation for 5 min at 3000 rpm and dried at 40 °C for 24 h.

The phase composition of prepared powders (cement solid phase and HAp) was analyzed using X-ray powder diffractometry (XRD, PANalytical X'Pert PRO, Westborough, MA). XRD patterns were recorded using Ni-filter and Cu Kα radiation at 40 kV and 30 mA, 2θ range of 5–60°. HAp suspension was dried at 100 °C for 24 h, followed by heat treatment in air atmosphere at 1100 °C for 1 h, before the XRD analysis. The specific surface area of α-TCP and as-synthesized HAp powder was determined using the BET method (ISO 9277:2010, Quadrasorb SI-KR/MP, Quantachrome Instruments, Boynton Beach, FL) measuring the amount of physically adsorbed N2 gas (purity 99.99%) at −196.15 °C. Before analysis all samples were degassed for 24 h at 100 °C. For the calculations the Brunauer–Emmett–Teller model has been applied. The value of specific surface area found was used to calculate the average α-TCP and as-synthesized HAp particle size, assuming that particles are spherical and the theoretical density of α-TCP = 2.86 g/cm3 and for HAp = 3.14 g/cm3 [6]. Calculations were done according to Eq. (2):

2.4. Preparation of vancomycin hydrochloride/PLA/HAp microcapsules PLA (1 g) was dissolved in 10 ml of dichloromethane. PVA (4 g) was dissolved in 100 ml of water. Vancomycin hydrochloride (1 g) was dissolved in 1 g of HAp suspension (cHAp = 0.2 g/ml). An aqueous suspension of VANK and HAp was added to the PLA solution in dichloromethane. S/W1/O (solid phase/primary water phase/organic phase) primary suspension was properly homogenized for 30 s at 7000 rpm and added to 100 ml of 4% aqueous PVA solution. S/W1/O/W2 double emulsion was homogenized for 30 s at 7000 rpm. After emulsification, the organic solvent was extracted in 2 L of water for 30 min. Then the microcapsules formed were separated by centrifugation for 5 min at 3000 rpm and dried at 40 °C for 24 h.

D¼

6 ; Sρ

ð2Þ

where D is the average particle diameter, S is the specific surface area and ρ is the theoretical density. Samples for mechanical tests were prepared by placing the CPC paste into cylindrical teflon molds (7.0 mm diameter × 16 mm height). After 24 h samples were removed from the teflon molds and the compressive strength was measured at a loading rate of 0.5 mm min− 1

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Table 1 Preparation of CPC samples. Composition

Liquid phase (μl)

Solid phase (mg)

Solid to liquid phase ratio (g/ml)

Vancomycin (mg)

PLA/vancomycin microcapsules (mg)

PLA/HAp/vancomycin microcapsules (mg)

CPC_1.89 CPC_1.75 CPC_1.23 CPC_1.89_vancomycin CPC_1.75_vancomycin CPC_1.23_vancomycin CPC_PLA/vancomycin CPC_PLA/HAp/vancomycin

370 400 570 370 400 570 570 570

700 700 700 700 700 700 700 700

1.89 1.75 1.23 1.89 1.75 1.23 1.23 1.23

– – – 15 15 15 – –

– – – – – – 350 –

– – – – – – – 350

using universal testing machine (INSTRON 10 kN, Norwood, MA). Five replicate samples were prepared and tested. Samples for the setting time evaluation were prepared by placing the CPC paste into cylindrical teflon molds (10 mm diameter × 5 mm height). The final setting time of all CPC compositions was determined at 21 °C using the standard Vicat needle method. Cement was considered to be set when no footprint of needle (1 mm in diameter and 270 g in weight) was observed. Three replicate samples were prepared and tested. The morphology and microstructure of CPC composites were observed from the fracture surface of the samples using scanning electron microscopy (SEM, Mira/LMU, Tescan, Brno, Czech Republic), at an acceleration voltage of 10 kV. Each sample was sputter coated with gold prior to imaging. The open (PO) porosity and total (PT) porosity of CPC were determined by the Archimedes method based on the principle, that buoyant force is equal to the weight of the fluid displaced [31]. To calculate PO and PT, the following equations were used: m1 −m0  100 m1 −m2 m0 ρAp ¼ m1 −m2 ρAp  100 P T ¼ 100− ρHAp P C ¼ P T −P O

PO ¼

molds (7.0 mm diameter × 16 mm height). After the preparation, samples were let to set for 24 h at 21 °C. Three replicate CPC composite samples from each batch were immersed in 20 ml of phosphate buffered saline (PBS), and incubated at 37 °C ± 0.5 °C and 50 rpm (Environmental Shaker — incubator ES-20, Biosan, Riga, Latvia). 2 ml aliquots of the solution were taken directly from the vessels after 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 24 h and once every following day for a period of 43 days. The volume taken was replaced with 2 ml of fresh PBS, keeping the total dissolution medium volume constant. Vancomycin content in dissolution medium was determined using ultraviolet–visible spectroscopy (UV/VIS spectrophotometer Evolution 300, Thermo Scientific, Waltham, MA) at λ = 280 nm.

2.9. Statistical evaluation All results were expressed as the mean value ± standard deviation (SD) of at least three independent samples. The significance of the results was evaluated using unpaired Student's t-test with the significance level set at p b 0.05. One-way and two-way analyses of variance (ANOVAs) were performed to evaluate the differences between the results.

3. Results 3.1. Preparation and evaluation of CPCs

where m1 — weight of impregnated porous sample, m0 — weight of dry porous sample, m2 — weight of impregnated porous sample in the water, ρAp — apparent density of porous sample, ρHAp — theoretical density of HAp (3.16 g/cm3) [17], and PC — closed porosity of porous sample. 2.8. Determination of drug release profiles in vitro Samples for the evaluation of in vitro vancomycin release from the CPC were prepared by placing the CPC paste into cylindrical teflon

XRD analysis (Fig. 1A) revealed that powders obtained by heating a mixture of CaCO3 and CaHPO4 (molar ratio 1:2) at 1300 °C for 4 h with subsequent quenching in air followed by milling in a planetary ball mill in isopropyl alcohol and used as calcium phosphate bone cement solid phase contain pure α-TCP phase (JCPDS 9-0348). During the research it was determined that α-TCP particles have irregular morphology (Fig. 1B), specific surface area of 1.5 g/m2 and average particle size (calculated from BET results) of 1.38 μm.

Fig. 1. Characterization of CPC solid phase: A) XRD patterns of α-tricalcium phosphate powder and B) SEM microphotograph of α-tricalcium phosphate powder.

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3.2. Preparation and characterization of CPC composites modified with õmicroencapsulated vancomycin

Table 2 Impact of solid to liquid phase ratio on the properties of CPC. Properties

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Solid to liquid phase ratio (g/ml)

Open porosity ± SD (%) Final setting time ± SD (min) Compression strength ± SD (MPa)

1.89

1.75

1.23

44.2 ± 0.2 17.5 ± 1.2 9.61 ± 2.31

46.1 ± 0.1 37.1 ± 1.6 6.82 ± 1.09

49.9 ± 0.1 51.4 ± 0.9 4.51 ± 0.38

In order to evaluate the liquid phase influence on the calcium phosphate bone cement final setting time, open porosity and mechanical properties, CPC samples with solid to liquid phase ratios of 1.89 g/ml, 1.75 g/ml and 1.23 g/ml were prepared. Results are summarized in Table 2. Obtained results indicated that by increasing the volume of liquid phase from 370 μl to 570 μl, open porosity of the samples increased, decreasing the mechanical properties of calcium phosphate bone cements from 9.61 ± 2.31 MPa down to 4.51 ± 0.38 MPa, respectively. The final setting time of the CPC samples was also strongly influenced by the volume of liquid phase used in the sample preparation process and increased almost 3 times if solid to liquid phase ratio was decreased from 1.89 g/ml to 1.23 g/ml. Cross section of CPC sample is shown in Fig. 2. The effect of the solid to liquid phase ratio on the vancomycin release kinetics as well as on the vancomycin burst release from the prepared CPC samples was evaluated (Fig. 3). Obtained results showed that with an increase of liquid phase volume the burst release of vancomycin within the first 24 h increased from 40.0 ± 2.1% (1.89 g/ml) up to 57.8% ± 1.2% (1.23 g/ml) (Fig. 3B). The impact of liquid phase amount on the vancomycin release was observed until the 8th day of experiment, when 90% of drug was transferred into the dissolution media (Fig. 3A). During the next 96 h, the rest of active substance was completely released.

In order to sustain vancomycin release from CPC for more than 12 days, the drug was encapsulated in the PLA matrix. Additionally nanosized hydroxyapatite particles were used in the encapsulation process to modify the surface and properties of obtained PLA/vancomycin microcapsules. Before the microencapsulation, it was determined that HAp powder obtained via wet precipitation reaction between calcium hydroxide and orthophosphoric acid contains pure HAp phase (JCPDS 9-0432, Fig. 4A), and HAp particles are needle shaped (Fig. 4B) with a specific surface area of 80 g/m2 and average particle size (calculated from BET results) of 24 nm. Analysis of the prepared microcapsules showed that upon encapsulation of vancomycin in the PLA matrix it is possible to obtain microcapsules in which the total vancomycin content equals to 4 wt.%, the repeatability of the microencapsulation process is ±3%, the encapsulation efficiency of the active ingredient reaches 40%, and the microcapsule sizes are in the range from 2 to 50 μm. In the case of PLA/HAp/ vancomycin microcapsules it has been found that the total vancomycin content in microcapsules equals to 4 wt.%, the repeatability of the microencapsulation process is ± 2%, the encapsulation efficiency of the active ingredient reaches 55% and the microcapsule sizes are in the range from 4 to 75 μm. The particle size distribution of prepared microcapsules is shown in Table 3. SEM investigations revealed that PLA/vancomycin microcapsules have a round shape and smooth surface (Fig. 5A). If HAp was used in the microencapsulation process, it was possible to obtain microcapsules not only loaded with vancomycin and HAp, but also coated with HAp nanoparticles (Fig. 5B). According to the morphology, obtained microcapsules can be classified as mono-cored or one domain microcapsules (Fig. 5C).

Fig. 2. SEM microphotographs: A and B) microstructure of CPC (solid to liquid phase ratio of 1.23 g/ml).

Fig. 3. Effect of the solid to liquid phase ratio on the: A) total vancomycin release and B) vancomycin initial burst release in the first 24 h.

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Fig. 4. Characterization of HAp powder: A) XRD patterns of HAp and B) SEM microphotograph of HAp particles.

4. Discussion Table 3 Particle size distribution of PLA/vancomycin and PLA/HAp/vancomycin microcapsules. Particle size distribution (μm) Microcapsules

d10

d50

d90

PLA/vancomycin microcapsules PLA/vancomycin/HAp microcapsules

4.19 6.96

13.24 24.20

34.84 53.12

Range of particle size (μm)

2–50 4–75

During the CPC modification with prepared microcapsules, it was found that the solid to liquid phase ratio of 1.89 g/ml is not appropriate to obtain injectable CPC paste, hence the solid to liquid phase ratio of 1.23 g/ml was used in order to obtain cement formulations that exhibited comparable injectability. Results showed that the burst release of vancomycin from the CPC/microcapsule composites was significantly decreased and only 7.7 ± 0.6% and 25.6 ± 4.0% of drug were released within the first 24 h in the case of PLA/vancomycin and PLA/HAp vancomycin microcapsules, respectively (Fig. 6B). Moreover, vancomycin release was sustained for more than 1032 h, if CPC/microcapsule composites (Fig. 7) were used, compared to 288 h, if drug was directly incorporated into the CPC matrix. Also the significant (p b 0.05) differences in total drug release were observed between CPCs modified with PLA/vancomycin (30.4 ± 1.3% released within 1032 h) and PLA/HAp/vancomycin (85.3 ± 3.1% released within 1032 h) microcapsules (Fig. 6A). During the study it was found that the addition of microcapsules to CPC significantly changed the final setting time of prepared composites and it decreased from 51.4 ± 0.9 min to 9.6 ± 1.2 min and 9.9 ± 1.6 min if CPC was modified with PLA/vancomycin or PLA/HAp/vancomycin microcapsules, respectively (Fig. 8). Significant differences between the microcapsule type on the CPC composite final setting time was not observed. CPC modification with both pure drug and vancomycin containing microcapsules decreased the cement mechanical properties by more than 27%. Significant differences between CPC modification with pure drug or vancomycin loaded microcapsules were not observed.

Usually infections caused by surgical procedures or bone infections such as osteomyelitis are treated using such antibiotics like gentamicin or vancomycin [32,33]. More often in clinical praxis the systemic antibiotic treatment is combined with the local drug delivery to reach the most optimal effect. Traditionally polymethylmethacrylate spheres (PMMA) loaded with gentamicin sulfate have been implanted in the infection site [34,35]. However, these materials are nonresorbable and must be removed after some months as well as during the setting reaction when they emit a large amount of heat, causing a risk of bone necrosis. An alternative approach is to use calcium phosphate bone cements, prepared by a combination of one or more calcium orthophosphates, which upon mixing with a liquid phase, usually water or an aqueous solution, form a paste which set and harden after being implanted within the body [36]. Although CPCs are injectable and their incorporation within the body can be performed through minimally invasive surgical technologies, still the possible bacterial contamination causes multiple risks responsible for the repeated surgery or even the failure of cemented materials, which can be prevented by loading the CPCs with antibiotics. In the current study α-TCP (Fig. 1) was chosen as the cement solid phase. Hence the setting reaction of α-TCP is too slow for the clinical applications; phosphate salt solution was used to obtain the cement setting time of 17.5 ± 1.2 min (Table 2). The particle size and surface area of the solid phase are mainly responsible for the cement final properties [37,38], therefore to decrease the particle size and increase the specific surface area, α-TCP was milled in isopropyl alcohol for 1 h. The main advantages of CPC, besides their bioactivity, are the possibility to adjust such cement properties as injectability, setting time, moldability, resorbability and drug release capability in order to obtain the end product with desirable characteristics. One of the ways to modify the properties of CPCs is the variations of solid to liquid phase ratio during the CPC preparation process [33,39]. In the current study the impact of solid to liquid phase ratio on the bone cement final setting time, porosity, mechanical properties and vancomycin release capability, including the impact on the initial burst release of

Fig. 5. SEM microphotographs of: A) PLA/vancomycin microcapsule; B) PLA/HAp/vancomycin microcapsule; and C) cross section of PLA/HAp/vancomycin microcapsule.

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Fig. 6. Effect of microcapsule type on the: A) total vancomycin release from CPC composites and B) vancomycin initial burst release in the first 24 h from CPC composites.

drug, was evaluated. As expected, the decrease of solid to liquid phase ratio resulted in longer final setting times and the mechanical properties of cements decreased with a decrease of solid to liquid phase ratio which can be strongly attributed to the formed cement porosity after the setting reaction (Table 2). It is highly important to evaluate the initial burst release of the drug, because the excessive concentrations of the drug in the burst phase may be toxic [40]. In the current research it has been shown that the solid to liquid phase ratio does not influence the total drug release and the complete vancomycin release is observed already after 12 days (Fig. 3A). At the same time variations of solid to liquid phase ratio strongly influenced the initial burst release of drug in the first 24 h (Fig. 3B). In analyzing the drug release profile, it can be assumed that immediately after the cement injection within the body most of the vancomycin will be released during the first four days, preventing the possible infections caused by surgical procedure, followed by a gradual release of drug for next 8 days ensuring the prophylactic effect. For the treatment of serious bone infections like osteomyelitis, systemic antibiotic course for up to 6 weeks is required. In order to reach the dual effect — to sustain the drug release for more than 12 days and to introduce the macroporosity in the calcium phosphate bone cement samples facilitating newly formed bone ingrowth [23, 25], microencapsulated vancomycin form (PLA/vancomycin microcapsules) was prepared (Fig. 5A). Using the W/O/W microencapsulation technique, particles where the drug is dispersed as a separate phase in the microcapsules were obtained. The drug release from such microcapsules is restricted by the network of channels connected to the particle surface, and the drug molecules close to the microcapsule surface determines the initial burst release, which in most cases should be lower if compared to bone cements modified with pure drug. Accordingly, the results obtained (Fig. 6B) confirmed that the initial burst release of vancomycin from the CPC/microcapsule composites was more than 7 times lower (7.7 ± 0.6% within first 24 h) than that of pure drug loaded cements (57.8 ± 1.2% within first 24 h). Analyzing previously reported drug release profiles from the CPC/microcapsule composites, it can be seen that drug release can be prolonged up to

60 days, but in most cases only 30–70% of the incorporated drug is released during this period. Also in the current research it has been found that drug release was significantly prolonged (from 12 days to more than 43 days), if CPCs were modified with PLA/vancomycin microcapsules, but only 30.4 ± 1.3% of vancomycin from the composites was released within 43 days (Fig. 6A). In order to increase the drug bioavailability, an attempt was made to modify the PLA/vancomycin microcapsule morphology [41,42]. For this purpose nanosized hydroxyapatite was chosen. The impact of hydroxyapatite particle addition to the CPCs has been described previously and it has been shown that CPC seeding with a HAp weight proportion greater than 1% can improve the reactivity of bone cement [43,44]. By slight modifications of the W/O/W microencapsulation technique, PLA/HAp/vancomycin microcapsules not only loaded with nanosized HAp and vancomycin but also coated with nanosized HAp particles were prepared. To our knowledge such microcapsules were obtained for the first time. In analyzing the vancomycin release profiles from the CPC composites modified with PLA/HAp/vancomycin microcapsules, it has been found that the initial burst release within the first 24 h (25.6 ± 4.0%) was in between the initial burst release of CPC modified with pure drug (57.8 ± 1.2%) and CPC composites modified with PLA/vancomycin microcapsules (7.7 ± 0.6%). Within 43 days already 85.3 ± 3.1% of vancomycin was released from the CPC composites modified with PLA/vancomycin microcapsules, certifying our presumption that modification of microcapsule morphology can provide higher drug bioavailability. We suppose that these differences in drug release profiles can be attributed to microcapsule morphology changes and we suspect that the porosity of microcapsules is mainly responsible for this effect. To confirm our suggestions further experiments will be performed. It is well known that besides the drug release kinetics, the addition of drug or microparticles to the CPC can affect the final setting time and mechanical properties of bone cements. It had been reported that the final setting time could be fastened with the addition of different types of microcapsules or microspheres to the CPC, at the same time preserving the mechanical properties practically unchanged in the

Fig. 7. SEM microphotographs of CPC/microcapsule composite.

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Fig. 8. Mechanical properties and final setting time of prepared CPCs (solid to liquid phase ratio of 1.23 g/ml).

first 24 h after the cement setting [26,45]. Results obtained showed the significant decrease in the compressive strength between unmodified and modified CPCs, but among the modifications significant differences in the compressive strength were not detected. Also the dramatic decrease of bone cement final setting time from 51.4 ± 0.9 min to 9.6 ± 1.2 and 9.9 ± 1.6 min was observed if CPCs were modified with PLA/vancomycin and PLA/HAp/vancomycin microcapsules, respectively. 5. Conclusions Solid to liquid phase ratio used in the preparation of calcium phosphate bone cements should be seriously evaluated, because it affects not only the CPC intrinsic properties like porosity, final setting time and compressive strength of final product, but also has a strong influence on the initial burst release of vancomycin in the first 24 h. CPC modification with vancomycin loaded PLA microcapsules decreased the initial burst release of drug for more than 7 times, while only 30.4 ± 1.3% of drug was released within 43 days. A new approach was found in order to increase the drug bioavailability. Modification of CPC with PLA/vancomycin microcapsules loaded and coated with nanosized hydroxyapatite resulted in 85.3 ± 3.1% of vancomycin release within 43 days. Acknowledgment This work has been supported by Riga Technical University within the project No. ZP-2013/19 and by the National Research Programme No. 2014.10-4/VPP-3/21 “MultIfunctional Materials and composItes, photonicS and nanotechnology (IMIS2)” Project No. 4 “Nanomaterials and nanotechnologies for medical applications”. References [1] D.W. Grainger, Targeted delivery of therapeutics to bone and connective tissues, Adv. Drug Deliv. Rev. 64 (2012) 1061–1062. [2] S. Bose, S. Tarafder, Calcium phosphate ceramic systems in growth factor and drug delivery for bone tissue engineering: a review, Acta Biomater. 8 (2012) 1401–1421. [3] D.G. Arkfeld, E. Rubenstein, Quest for the Holy Grail to cure arthritis and osteoporosis: emphasis on bone drug delivery systems, Adv. Drug Deliv. Rev. 57 (2005) 939–944. [4] M.P. Ginebra, T. Traykova, J.A. Planell, Calcium phosphate cements as bone drug delivery systems: a review, J. Control. Release 113 (2006) 102–110. [5] S. Samavedi, A.R. Whittington, A.S. Goldstein, Calcium phosphate ceramics in bone tissue engineering: a review of properties and their influence on cell behavior, Acta Biomater. 9 (2013) 8037–8045. [6] S.V. Dorozhkin, Calcium orthophosphates, J. Mater. Sci. 42 (2007) 1061–1095. [7] M. Bohner, S. Tadier, N. Garderen, A. Gasparo, N. D belin, G. Baroud, Synthesis of spherical calcium phosphate particles for dental and orthopedic applications, Biomaterials 3 (2013) 1–15. [8] C. Canal, M.P. Ginebra, Fiber-reinforced calcium phosphate cements: a review, J. Mech. Behav. Biomed. Mater. 4 (2011) 1658–1671. [9] Z. Xie, X. Liu, W. Jia, C. Zhang, W. Huang, J. Wang, Treatment of osteomyelitis and repair of bone defect by degradable bioactive borate glass releasing vancomycin, J. Control. Release 139 (2009) 118–126.

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