Materials Science and Engineering C 37 (2014) 54–59
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Preparation and characterization of antimicrobial nano-hydroxyapatite composites Juhong Yu a, Xiaobing Chu b, Yurong Cai a, Peijian Tong b, Juming Yao a,⁎ a The Key Laboratory of Advanced Textile Materials and Manufacturing Technology of Ministry of Education, College of Materials and Textiles, Zhejiang Sci-Tech University, Hangzhou 310018, China b The First Affiliated Hospital, Zhejiang Chinese Medicine University, Hangzhou 310006, China
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Article history: Received 4 June 2013 Received in revised form 2 December 2013 Accepted 27 December 2013 Available online 3 January 2014 Keywords: Nano-hydroxyapatite Vancomycin hydrochloride Antibacterial Drug release
a b s t r a c t Deep infection of prosthesis is one of the most frequent complications after joint replacement. One of the most effective ways is to introduce directly some antibiotics in the local site of the surgery. In the present study, an antimicrobial composite has been fabricated using nano-hydroxyapatite particles as carriers for the antimicrobial drug of vancomycin hydrochloride (VAN) and the mixture of oxidation sodium alginate (OSA) and gelatin (GT) as a sticky matrix. Samples have been characterized using X-ray diffraction instrument (XRD), field emission scanning electron microscope (FE-SEM), transmission electron microscope (TEM) and Fourier transform infrared (FTIR) spectra, Brunauer–Emmett–Teller (BET) methods, the rotational rheometer and the texture analyzer. The release of VAN from nano-hydroxyapatite (nHAP) particles was detected by the ultraviolet–visible (UV–vis) spectrophotometer and then bactericidal property of the composite was evaluated using the Staphylococcus aureus (S. aureus) as a bacterial model. Experimental results showed that the composite possessed an adhesive property derived from the gel of OSA and GT, which implied that the composite could bond directly to the fracture surface of bones in surgery. Furthermore, VAN was loaded efficiently on the surface of nHAP particles and could be released slowly from these particles, which endowed the composite with an obvious and continuous antimicrobial performance. The sticky and antimicrobial composite may has a potential application in arthroplasty to overcome deep infection in a simple and direct manner. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Osteoarthritis (OA) is a chronic regional joint disease along with musculoskeletal pain and disability, which causes function impairment of limbs and reduction of life quality [1]. The joint replacement surgeries are the routine methods using the artificial materials to reshape a new joint of which the function is close to the normal one, relieving the pain and reducing the deformity and dysfunction of joint [2]. But deep infection [3] of prosthesis is one of the most challenging complications of total joint arthroplasty, which may lead to the failure of arthroplasty and the removal of implants. The most direct and effective method to solve this problem is to introduce some antibiotics in the local sites of surgery for the purpose of prophylaxis or treatment [4,5]. In fact, antibiotic-impregnated cement has been used widely in clinics of US hospitals for many years. According to the report of Parviz et al. [6], the use of antibiotic-impregnated cement lowered the infection rate by approximately 50% in primary hip arthroplasty. But in many cases, the antibiotic has been mixed simply into cement at the time of surgery, which causes some problems related to the poor drug release kinetics and unreasonable drug-loaded quantity [7,8]. The overuse of topical ⁎ Corresponding author. Tel.: +86 571 86843618; fax: +86 571 86843619. E-mail address: [email protected] (J. Yao). 0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.12.038
antibiotics causes other concerns related to antibiotic resistance and interference with the surgical site healing process etc. [9]. In attempts to combat infection, research and development of new biocompatible materials are appealed strongly with more convenient operation, more rational drug release kinetics and lower side effects. Hydroxyapatite (HAP, Ca10 (PO4)6(OH)2), the main inorganic component of the vertebrate animals' teeth and bones, has been widely used as the repairing/replacement material of hard tissue due to its properties of endophilicity, non-toxic, non-stimulating, non-allergenic, nonmutagenic and osteoconductive, etc. [10–12]. Also, nano-hydroxyapatite (nHAP) particles have been used as carriers of growth factor [13], antibiotics [14], anticancer drugs [15], and enzymes [16] for its notorious adsorption ability. Recently, we have successfully fabricated ibuprofen and/or vitamin C-loaded nHAP particles [17,18]. The nanoparticle exhibits an effective effect on the release of drug with a sustained rate. In the present work, nHAP particles have been designed as carriers of antimicrobial drug of vancomycin hydrochloride (VAN) to control its release. Before being used, the VAN-loaded nHAP particles have been mixed with the mixture of oxidized sodium alginate (SA) and gelatin (GT) [19,20] to obtain a sticky composite [21], with which VAN-loaded HAP particles could be fixed directly on the surface of broken bone and treated deep infections in the artificial joint replacement and bone tissue engineering through a simple procedure.
J. Yu et al. / Materials Science and Engineering C 37 (2014) 54–59
2. Experimental part 2.1. Materials VAN was purchased from Eli Lilly Japan K.K. Bombyx mori silk sericin (SS) protein (molecular weight, 8 kDa) was obtained from Huzhou Aotesi Biotechnology Co., Ltd., China. Sodium alginate (SA) was purchased from Maichao (Shanghai) Import & Export Trading Co., Ltd., China. Gelatin (GT) was provided by Shanghai Shengaming Biotechnology Co., Ltd., China. Staphylococcus aureus (S. aureus) was provided by Shanghai Institutes for Biological Sciences, China. NaIO4, CaCl2, NaOH, Na2HPO4, glycol, NaCl, absolute ethyl alcohol and borax were of analytical grade and purchased from Hangzhou Mike Chemical Instrument Co., Ltd., China. 2.2. Preparation of nHAP particles First, 1.25 g SS protein was completely dissolved in 150 mL ddH2O at 50 °C, followed by adding 50 mL CaCl2 (0.05 M) into the SS solution and stirring for 30 min. Fifty milliliters of Na2HPO4 (0.03 M) was then added dropwise into the SS–CaCl2 solution, while keeping the pH value of the reaction system at 10 by NaOH aqueous solution (1 M). After the reaction, the precipitates were collected by centrifugation and rinsed with ddH2O and absolute ethyl alcohol for 3 times alternatively [22,23]. The precipitates were lyophilized and sintered in a muffle furnace last at 650 °C for 3 h to obtain the nHAP particles.
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VAN-nHAP particles were then immersed in 5 mL simulated body fluid (SBF, pH 7.4) and the release of VAN was conducted in an incubator shaker at 37 °C. A 0.5 mL solution was withdrawn at the appropriate time points of 2, 4, 6, 8, 10, 12, 24, 48, 72, and 96 h for release observation of VAN. Meanwhile, 0.5 mL fresh SBF pre-warmed at 37 °C was added into the release medium to maintain its constant volume. The release amount of VAN at each time point was calculated according to the standard curve of VAN. 2.4. Preparation of nHAP/OSA/GT composites SA solution (2 (w/v)%) was obtained by dissolving 4.0 g SA in 200 mL ddH2O. NaIO4 (2.0 g) was then added into the SA solution with agitation at 25 °C for 4 h in the dark, followed by adding 2 mL glycol. After another 2 h, 0.5 g NaCl was dissolved in the reaction solution and 400 mL ethanol was added to precipitate the oxidation sodium alginate (OSA) [24], which were collected by centrifugation. The obtained OSA precipitates were redissolved in 100 mL ddH2O, followed by adding 0.50 g NaCl and 200 mL ethanol successively to precipitate the OSA again. This procedure was repeated 3 times in order to remove the impurities. Ten percent OSA and 15% GT solutions were prepared by dissolving OSA and GT in PBS (pH 7.4) respectively and their pH values were adjusted to 7.4 with 0.1 M borax. Ten percent OSA and 15% GT solutions were mixed to obtain a series of OSA/GT composites, in which the volume ratios of 10% OSA to 15% GT were 2:1, 1:2, 1:3 and 1:4 respectively. The VAN-nHAP/OSA/GT composites were prepared by adding VAN-nHAP particles into the OSA and GT mixed solutions with varied certain proportions.
2.3. Preparation and in vitro release of VAN-nHAP particles 2.5. Characterization A series of VAN solution with different concentrations were prepared, of which the absorbance was measured by the UV–vis spectrophotometer (Beckman Coulter, DU530) at 280 nm with 3 mL solution in the silica dish. The VAN standard curve was drawn based on the absorbance and VAN concentration. For the preparation of VAN-loaded nHAP particles, 5 mg VAN was dissolved in 2 mL of simulated body fluid (SBF, pH 7.4) first. One hundred milligrams of nHAP particles was then added into the VAN solution, and kept in a vacuum condition at ambient temperature for 24 h. The same amount of nHAP was added into 2 mL SBF in the absence of VAN as the control. The precipitates, i.e., VAN-nHAP particles, were collected by centrifugation and the residual amount of VAN in the supernatant was determined by measuring its concentration in supernatant with UV–vis spectrophotometer. Amount of VAN loaded on the nHAP particles was then calculated using the weight differential method. The load rate of VAN was calculated by the ratio of VAN amount loaded on the nHAP and the total quantity of VAN.
211 112
a
The chemical composition and crystallinity of nHAP particles were analyzed with X-ray diffraction instrument (XRD) (ARL X'TRA, Thermo Electron) using a monochromatic CuKα radiation (λ = 1.54056 nm) in a range of 2θ = 10°–70° with a rate of 5°/min and voltage of 40 kV, respectively. The morphology and size of nHAP particles were investigated using a transmission electron microscope (TEM) (JEM-1230, JEOL) at 80 kV after they were dispersed in the absolute ethyl alcohol. Brunauer–Emmett–Teller (BET) surface areas of the nHAP particles were determined by a surface area and porosity analyzer (F-Sorb 3400, Gold Application). The samples were heated at 110 °C for 2 h in a vacuum before the test in order to remove absorbed vapor and air. Three composites of OSA, GT and OSA/GT (1:2) were ground into the powder and mixed with KBr in a mass ratio of 1:100 for Fourier transform infrared (FTIR) observation, which were recorded on an attenuated total reflection FTIR instrument (Nicolet 5700, Thermo Electron) in the range of 400–4000 cm− 1 with a resolution of 4 cm−1.
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2θ/(°) Fig. 1. XRD pattern (a) and TEM image (b) of nano-hydroxyapatite (nHAP) particles.
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Vancomycin concentration (µg·ml-1)
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b Cumulative release concentration of VAN (%)
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y = 210.66x - 1.201 R2 = 0.9999
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Absorbance Fig. 2. The standard curve (a) and cumulative release curve (b) of VAN.
The oxidation degree of the OSA was measured by the potentiometric titration of hydroxylamine hydrochloride [25]. The rheological behaviors of several OSA/GT composites were studied using the rotational rheometer (AR-G2) (TA Instruments, USA), in which the diameter and interval of probes were 20 mm and 0.341 cm; constant temperature, pressure and frequency were 25 °C, 30 Pa and 1 Hz, respectively. The stickiness and stringiness of OSA/GT and VAN-nHAP/ OSA/GT composites were measured ten times per sample by the texture analyzer (TA.XT Plus) (SMS, UK). The dispersion of nHAP in OSA/GT mixture was detected using a field emission scanning electron microscope (FE-SEM) (S4800, Hitachi) with an accelerating voltage of 1 kV after the mixture was coated on the sample table and dried.
For in vitro antibacterial test, a 1 mL OSA/GT composite solution containing 50 mg VAN-nHAP particles was shaped as a columnar gel (ϕ2.2 cm×0.3 cm), in which the volume ratio of OSA to GT was 1:2. The sample without VAN was used as the control and named as nHAP/ OSA/GT. The columnar gels were placed on the culture dishes coated with S. aureus, which were cultured at 37 °C for 24 h for the detection of inhibition zone. Meanwhile, the columnar gels were soaked in 5 mL SBF, respectively. After 1, 3, 5 and 7 days of soaking, the supernatants were collected and mixed with S. aureus bacteria culture medium respectively in the proportion of 1:1 and coated on the culture dishes with 6 replicates per group. After 24 h of culture, the colony forming units (CFU) of S. aureus were counted by the enumeration method of colonies on plate to evaluate the sustained antibacterial effect of the composites.
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Fig. 3. FTIR spectra of SA, OSA, GT and OSA/GT (a), the hydroxylamine hydrochloride–potential titration curve (b) and the first differential curve (c) of OSA.
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3. Results and discussion 3.1. Characterization of nHAP The implant-related infection is one of the most common complications in arthroplasty. The routine method to prevent the deep infection is using antibiotic-impregnated cement. But the high dose and irrational release cycle of drugs may lead to many undesirable side effects. The ideal strategy is to introduce directly a drug delivery system in the local site of surgery, improving the anti-infection properties for a long time. Hence, self-made nanoparticles had been used as carriers for the treatment of infection. First, chemical composition of the nanoparticles has been characterized using XRD and the predominant HAP crystal phase had been confirmed according to the standard card of HAP (JCPDS 09-432) (as shown in Fig. 1a). The peaks in the pattern were sharp, which implied that the nanoparticles had higher crystallinity.
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The morphology of nHAP was shown in Fig. 1b, in which the particles had an irregular shape and their sizes were ca. 30–40 nm. Some of the particles adhered to each other because of the fusion of crystal during the sintering process. According to the BET method, the specific surface area, pore diameter and porosity of nHAP were analyzed and results were 67.82 m2 g−1, 11.72 nm and 51.74%, respectively. Usually, drug adsorption happened mainly on the surface of adsorbent. So the nHAP particles with bigger specific surface area and higher porosity were conducive to the drug loading in subsequent experiments. 3.2. Load and release of VAN In order to determine the drug loading-release property and bactericidal property of the nHAP particles, VAN has been used as a model drug. The drug loading process has been performed by soaking the nHAP particles in SBF solution containing VAN. The load capacity of
Fig. 4. The rheogram of four OSA/GT samples: a: viscosity–time curves; b: G', G″–time curves.
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a1
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Fig. 5. FE-SEM images of OSA/GT (a1) and VAN-nHAP/OSA/GT (a2) composites and digital photos of OSA/GT (b1) and VAN-nHAP/OSA/GT (b2) by the method of TA.XT Plus.
VAN on every 100 mg nHAP was measured using the weight difference method on the base of the curve of VAN content and absorbance (Fig. 2a). VAN (955.68 ± 25.44 μg) has been loaded on every 100 mg nHAP particles and load rate was up to 19.11 ± 0.29%. Based on the cumulative release curve in Fig. 2b, the release process of VAN could be divided into two parts according to its release rate. There was a burst release of VAN at the first 24 h, which reached 61% of total amount of loaded VAN on nHAP, which could be ascribed to VAN adsorbed on the surface of nHAP. The release rate reduced gradually after the initial release phase. Released drug in the second process could be attributed to VAN encapsulated in the porous channels. About 72% of VAN loaded on nHAP was released after 375 h and the release was also last with the increase of immersing time, which suggested that nHAP could be served as an effective carrier to modulate the release of VAN with a slower release rate and a longer release time. 3.3. Characterization of OSA/GT and VAN-nHAP/OSA/GT composites When used in the fracture surface of bones, an ideal carrier material for the treatment of bone infection should not only possess a reasonable drug loading-releasing property but also be fixed in a close site to fracture bone so as to maximize the function of the antibiotic. Hence, a sticky matrix composed of OSA and GT had been constructed to bond VNA-loaded nHAP particles to the bone fracture surface. Sodium periodate, as a strong oxidizing agent, can oxidize the adjacent glycol structure of SA into the adjacent dialdehyde group [26]. The aldehyde group of OSA was demonstrated by the result of FTIR (Fig. 3a). SA, which contains a lot of hydroxyl, has strong polarity and can form hydrogen bonds with electron donors (such as –NH2, –OH, C–O–C) which have lone pair electrons. When hydrogen bond is formed in the SA matrix, stretching vibration frequency of –OH will decrease and the bond shape will become wider [27]. In Fig. 3a, there was a strong and wide diffraction peak of ν(O–H) at 3428.4 cm−1 and a stretching vibration diffraction peak of C–O at 1031.6 cm−1 in the spectrum of SA. Compared with the spectrum of SA, a new characteristic diffraction peak appeared in the spectrum of OSA at 1799.6 cm−1, which could be ascribed to the vibration absorption peak of C = O of the aldehyde group. Along with the oxidation process, the hydrogen bonds were weakened and the stretching vibration frequency of –OH moved from 3428.4 cm−1 to 3446.2 cm−1 and the bond was narrower, which suggested that the aldehyde group of OSA was obtained during the periodate oxidation of SA. According to the hydroxylamine hydrochloride–potential titration curve (Fig. 3b) and its first differential curve (Fig. 3c) of OSA, the aldehyde group concentration ([CHO], mol/g) and degree of oxidation (OD) were calculated, which were 5.491 ± 0.008 mmol/g and 54.39 ± 0.08% respectively. Schiff base can be formed by the reaction of the aldehyde group of OSA and amino of GT. The FTIR spectra of OSA, GT and OSA/GT (Fig. 3a) showed that, the characteristic diffraction peaks of aldehyde group of OSA at 1799.6 cm−1 and amide related to GT at 1653.4 and 1551.3 cm− 1 were weakened in the OSA/GT composite due to the formation of the Schiff base. Besides, there was a strong characteristic
diffraction peak of C=N bond at 1643.0 cm−1, which could be assigned to the characteristic absorption peak of Schiff base [28], and the peak became wider because of the overlapping with the absorption peak at 1653.4 cm−1 of GT. All results showed that Schiff base was obtained due to the crosslinking of OSA and GT. In order to understand their gel transition times, the rheological behaviors of four OSA/GT composites with different ratio of OSA and GT were measured (Fig. 4). The dynamic storage modulus (G') reflects the elasticity of the system, which will overlap with the dynamic loss modulus (G") when the crosslinking system is close to the gel point. So the time of G'=G" can be defined as gel time [29]. In this research, the concentration of OSA and GT selected was 10% and 15% respectively. The viscosity–time curves of OSA/GT with different volume ratios (Fig. 4a) showed that, the viscosity of the system increased with the increase of time. Meanwhile, the viscosity of composite increased with the increase of the volume of GT. The G', G"–time curves of the OSA/GT systems (Fig. 4b) showed the gel times of these composites were 2160 s, 822 s, 330 s and 268 s when the ratios of OSA to GT were 2:1, 1:2, 1:3 and 1:4 respectively, which indirectly implied the change trend of their viscosity over time. Considering the rheological results and operation convenience in practical application, the volume ratio of 10% OSA and 15% GT selected for antibacterial experiment was 1:2. Furthermore, the surfaces of glass plates were coated with both OSA/GT and VAN-nHAP/OSA/GT composites respectively to observe the distribution of VAN-nHAP particles in the composite using FE-SEM (Fig. 5a1–a2). VAN-nHAP particles could be dispersed homogeneously in the composite, which was beneficial to stable and homogeneous release of VAN from the composite. Both composites of OSA/GT and VAN-nHAP/OSA/GT had been measured by the texture analyzer. Unlike the translucence of OSA/GT, VAN-nHAP/OSA/GT composite was ivory due to the presence of VAN-nHAP nanoparticles according to the photographs of composites (Fig. 5b1–b2). Moreover, both composites showed an obvious adhesive ability between operation panel and probe. The specific data of hardness, stickiness and stringiness were obtained and results presented here revealed better reproducibility among replicates in Table 1. Both OSA/GT and VAN-nHAP/OSA/GT composites had certain and similar stickiness. Obviously, the presence of VAN-nHAP particles in OSA/GT composite had not changed evidently gel's stickiness. By the stickiness of OSA/GT composite, VAN-nHAP particles could be fixed directly on the fracture surface of bone. As shown in Fig. 6a, compared with nHAP/OSA/GT, the VAN-nHAP/ OSA/GT composite had an obvious bacteriostatic ring, indicating that the VAN-nHAP/OSA/GT composite has a certain bacteriostatic effect. Fig. 6b showed the sustained antibacterial effect of the composites. It can be seen that there is no significant difference between the colony Table 1 The result of TA.XT Plus of composites. Sample
Hardness (g)
Stickiness (g)
Stringiness (mm)
OSA/GT VAN-nHAP/OSA/GT
13.885 ± 0.002 14.208 ± 0.003
4.411 ± 0.005 5.591 ± 0.003
7.153 ± 0.010 7.159 ± 0.008
J. Yu et al. / Materials Science and Engineering C 37 (2014) 54–59
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Fig. 6. Results of inhibition zone test and colony count of samples (S. aureus). ⁎⁎p b 0.01.
count of blank control and nHAP/OSA/GT composites, which indicated that samples in the absence of VAN had little bacteriostatic effect. The introduction of VAN into the composites brought a significant decrease (p b 0.01) of the colony count, which implied that VAN-nHAP/OSA/GT composite had an excellent antibacterial effect. With the increase of culturing time, the colony count of blank control and nHAP/OSA/GT composite had no obvious change. However, the colony count of VAN-nHAP/ OSA/GT composite continually decreased from (10.3 ± 0.2) × 103 to (9.96 ± 0.08) × 103, (9.5 ± 0.2) × 103 and (9.1 ± 0.1) × 103 cfu mL−1 with the increase of the culture time from 1 to 3, 5 and 7days respectively. The results demonstrated that the VAN-nHAP/OSA/GT composite had a continuous enhancement of antibiotic resistance. This was mainly attributed to the slow-release of VAN from the nHAP particles, which was consistent with the result of Fig. 2b. 4. Conclusion VAN-loaded HAP nanoparticles have been fabricated and the nanoparticles showed a higher drug loading efficiency and better drug controlled release property. After VAN-loaded nHAP combined with OSA/GT gel, viscous VAN-nHAP/OSA/GT composite was obtained. The composite possessed an adhesive property derived from the gel of OSA and GT, and an obvious long-term and continuous antimicrobial performance derived from VAN-loaded nHAP particles. The sticky and antimicrobial composite may has a potential application in arthroplasty to overcome deep infection and improve the success rate of surgery in a simple and direct manner. Acknowledgement The work is financially supported by the Program for National Natural Science Foundation of China (51372226, 51172207, 51202219) and Qianjiang Talent Project of Zhejiang Province (2013R10062).
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Preparation and characterization of antimicrobial nano-hydroxyapatite composites Juhong Yu a, Xiaobing Chu b, Yurong Cai a, Peijian Tong b, Juming Yao a,⁎ a The Key Laboratory of Advanced Textile Materials and Manufacturing Technology of Ministry of Education, College of Materials and Textiles, Zhejiang Sci-Tech University, Hangzhou 310018, China b The First Affiliated Hospital, Zhejiang Chinese Medicine University, Hangzhou 310006, China
a r t i c l e
i n f o
Article history: Received 4 June 2013 Received in revised form 2 December 2013 Accepted 27 December 2013 Available online 3 January 2014 Keywords: Nano-hydroxyapatite Vancomycin hydrochloride Antibacterial Drug release
a b s t r a c t Deep infection of prosthesis is one of the most frequent complications after joint replacement. One of the most effective ways is to introduce directly some antibiotics in the local site of the surgery. In the present study, an antimicrobial composite has been fabricated using nano-hydroxyapatite particles as carriers for the antimicrobial drug of vancomycin hydrochloride (VAN) and the mixture of oxidation sodium alginate (OSA) and gelatin (GT) as a sticky matrix. Samples have been characterized using X-ray diffraction instrument (XRD), field emission scanning electron microscope (FE-SEM), transmission electron microscope (TEM) and Fourier transform infrared (FTIR) spectra, Brunauer–Emmett–Teller (BET) methods, the rotational rheometer and the texture analyzer. The release of VAN from nano-hydroxyapatite (nHAP) particles was detected by the ultraviolet–visible (UV–vis) spectrophotometer and then bactericidal property of the composite was evaluated using the Staphylococcus aureus (S. aureus) as a bacterial model. Experimental results showed that the composite possessed an adhesive property derived from the gel of OSA and GT, which implied that the composite could bond directly to the fracture surface of bones in surgery. Furthermore, VAN was loaded efficiently on the surface of nHAP particles and could be released slowly from these particles, which endowed the composite with an obvious and continuous antimicrobial performance. The sticky and antimicrobial composite may has a potential application in arthroplasty to overcome deep infection in a simple and direct manner. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Osteoarthritis (OA) is a chronic regional joint disease along with musculoskeletal pain and disability, which causes function impairment of limbs and reduction of life quality [1]. The joint replacement surgeries are the routine methods using the artificial materials to reshape a new joint of which the function is close to the normal one, relieving the pain and reducing the deformity and dysfunction of joint [2]. But deep infection [3] of prosthesis is one of the most challenging complications of total joint arthroplasty, which may lead to the failure of arthroplasty and the removal of implants. The most direct and effective method to solve this problem is to introduce some antibiotics in the local sites of surgery for the purpose of prophylaxis or treatment [4,5]. In fact, antibiotic-impregnated cement has been used widely in clinics of US hospitals for many years. According to the report of Parviz et al. [6], the use of antibiotic-impregnated cement lowered the infection rate by approximately 50% in primary hip arthroplasty. But in many cases, the antibiotic has been mixed simply into cement at the time of surgery, which causes some problems related to the poor drug release kinetics and unreasonable drug-loaded quantity [7,8]. The overuse of topical ⁎ Corresponding author. Tel.: +86 571 86843618; fax: +86 571 86843619. E-mail address: [email protected] (J. Yao). 0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.12.038
antibiotics causes other concerns related to antibiotic resistance and interference with the surgical site healing process etc. [9]. In attempts to combat infection, research and development of new biocompatible materials are appealed strongly with more convenient operation, more rational drug release kinetics and lower side effects. Hydroxyapatite (HAP, Ca10 (PO4)6(OH)2), the main inorganic component of the vertebrate animals' teeth and bones, has been widely used as the repairing/replacement material of hard tissue due to its properties of endophilicity, non-toxic, non-stimulating, non-allergenic, nonmutagenic and osteoconductive, etc. [10–12]. Also, nano-hydroxyapatite (nHAP) particles have been used as carriers of growth factor [13], antibiotics [14], anticancer drugs [15], and enzymes [16] for its notorious adsorption ability. Recently, we have successfully fabricated ibuprofen and/or vitamin C-loaded nHAP particles [17,18]. The nanoparticle exhibits an effective effect on the release of drug with a sustained rate. In the present work, nHAP particles have been designed as carriers of antimicrobial drug of vancomycin hydrochloride (VAN) to control its release. Before being used, the VAN-loaded nHAP particles have been mixed with the mixture of oxidized sodium alginate (SA) and gelatin (GT) [19,20] to obtain a sticky composite [21], with which VAN-loaded HAP particles could be fixed directly on the surface of broken bone and treated deep infections in the artificial joint replacement and bone tissue engineering through a simple procedure.
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2. Experimental part 2.1. Materials VAN was purchased from Eli Lilly Japan K.K. Bombyx mori silk sericin (SS) protein (molecular weight, 8 kDa) was obtained from Huzhou Aotesi Biotechnology Co., Ltd., China. Sodium alginate (SA) was purchased from Maichao (Shanghai) Import & Export Trading Co., Ltd., China. Gelatin (GT) was provided by Shanghai Shengaming Biotechnology Co., Ltd., China. Staphylococcus aureus (S. aureus) was provided by Shanghai Institutes for Biological Sciences, China. NaIO4, CaCl2, NaOH, Na2HPO4, glycol, NaCl, absolute ethyl alcohol and borax were of analytical grade and purchased from Hangzhou Mike Chemical Instrument Co., Ltd., China. 2.2. Preparation of nHAP particles First, 1.25 g SS protein was completely dissolved in 150 mL ddH2O at 50 °C, followed by adding 50 mL CaCl2 (0.05 M) into the SS solution and stirring for 30 min. Fifty milliliters of Na2HPO4 (0.03 M) was then added dropwise into the SS–CaCl2 solution, while keeping the pH value of the reaction system at 10 by NaOH aqueous solution (1 M). After the reaction, the precipitates were collected by centrifugation and rinsed with ddH2O and absolute ethyl alcohol for 3 times alternatively [22,23]. The precipitates were lyophilized and sintered in a muffle furnace last at 650 °C for 3 h to obtain the nHAP particles.
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VAN-nHAP particles were then immersed in 5 mL simulated body fluid (SBF, pH 7.4) and the release of VAN was conducted in an incubator shaker at 37 °C. A 0.5 mL solution was withdrawn at the appropriate time points of 2, 4, 6, 8, 10, 12, 24, 48, 72, and 96 h for release observation of VAN. Meanwhile, 0.5 mL fresh SBF pre-warmed at 37 °C was added into the release medium to maintain its constant volume. The release amount of VAN at each time point was calculated according to the standard curve of VAN. 2.4. Preparation of nHAP/OSA/GT composites SA solution (2 (w/v)%) was obtained by dissolving 4.0 g SA in 200 mL ddH2O. NaIO4 (2.0 g) was then added into the SA solution with agitation at 25 °C for 4 h in the dark, followed by adding 2 mL glycol. After another 2 h, 0.5 g NaCl was dissolved in the reaction solution and 400 mL ethanol was added to precipitate the oxidation sodium alginate (OSA) [24], which were collected by centrifugation. The obtained OSA precipitates were redissolved in 100 mL ddH2O, followed by adding 0.50 g NaCl and 200 mL ethanol successively to precipitate the OSA again. This procedure was repeated 3 times in order to remove the impurities. Ten percent OSA and 15% GT solutions were prepared by dissolving OSA and GT in PBS (pH 7.4) respectively and their pH values were adjusted to 7.4 with 0.1 M borax. Ten percent OSA and 15% GT solutions were mixed to obtain a series of OSA/GT composites, in which the volume ratios of 10% OSA to 15% GT were 2:1, 1:2, 1:3 and 1:4 respectively. The VAN-nHAP/OSA/GT composites were prepared by adding VAN-nHAP particles into the OSA and GT mixed solutions with varied certain proportions.
2.3. Preparation and in vitro release of VAN-nHAP particles 2.5. Characterization A series of VAN solution with different concentrations were prepared, of which the absorbance was measured by the UV–vis spectrophotometer (Beckman Coulter, DU530) at 280 nm with 3 mL solution in the silica dish. The VAN standard curve was drawn based on the absorbance and VAN concentration. For the preparation of VAN-loaded nHAP particles, 5 mg VAN was dissolved in 2 mL of simulated body fluid (SBF, pH 7.4) first. One hundred milligrams of nHAP particles was then added into the VAN solution, and kept in a vacuum condition at ambient temperature for 24 h. The same amount of nHAP was added into 2 mL SBF in the absence of VAN as the control. The precipitates, i.e., VAN-nHAP particles, were collected by centrifugation and the residual amount of VAN in the supernatant was determined by measuring its concentration in supernatant with UV–vis spectrophotometer. Amount of VAN loaded on the nHAP particles was then calculated using the weight differential method. The load rate of VAN was calculated by the ratio of VAN amount loaded on the nHAP and the total quantity of VAN.
211 112
a
The chemical composition and crystallinity of nHAP particles were analyzed with X-ray diffraction instrument (XRD) (ARL X'TRA, Thermo Electron) using a monochromatic CuKα radiation (λ = 1.54056 nm) in a range of 2θ = 10°–70° with a rate of 5°/min and voltage of 40 kV, respectively. The morphology and size of nHAP particles were investigated using a transmission electron microscope (TEM) (JEM-1230, JEOL) at 80 kV after they were dispersed in the absolute ethyl alcohol. Brunauer–Emmett–Teller (BET) surface areas of the nHAP particles were determined by a surface area and porosity analyzer (F-Sorb 3400, Gold Application). The samples were heated at 110 °C for 2 h in a vacuum before the test in order to remove absorbed vapor and air. Three composites of OSA, GT and OSA/GT (1:2) were ground into the powder and mixed with KBr in a mass ratio of 1:100 for Fourier transform infrared (FTIR) observation, which were recorded on an attenuated total reflection FTIR instrument (Nicolet 5700, Thermo Electron) in the range of 400–4000 cm− 1 with a resolution of 4 cm−1.
b
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222 213
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nHAP
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2θ/(°) Fig. 1. XRD pattern (a) and TEM image (b) of nano-hydroxyapatite (nHAP) particles.
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Vancomycin concentration (µg·ml-1)
200
90
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b Cumulative release concentration of VAN (%)
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y = 210.66x - 1.201 R2 = 0.9999
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Absorbance Fig. 2. The standard curve (a) and cumulative release curve (b) of VAN.
The oxidation degree of the OSA was measured by the potentiometric titration of hydroxylamine hydrochloride [25]. The rheological behaviors of several OSA/GT composites were studied using the rotational rheometer (AR-G2) (TA Instruments, USA), in which the diameter and interval of probes were 20 mm and 0.341 cm; constant temperature, pressure and frequency were 25 °C, 30 Pa and 1 Hz, respectively. The stickiness and stringiness of OSA/GT and VAN-nHAP/ OSA/GT composites were measured ten times per sample by the texture analyzer (TA.XT Plus) (SMS, UK). The dispersion of nHAP in OSA/GT mixture was detected using a field emission scanning electron microscope (FE-SEM) (S4800, Hitachi) with an accelerating voltage of 1 kV after the mixture was coated on the sample table and dried.
For in vitro antibacterial test, a 1 mL OSA/GT composite solution containing 50 mg VAN-nHAP particles was shaped as a columnar gel (ϕ2.2 cm×0.3 cm), in which the volume ratio of OSA to GT was 1:2. The sample without VAN was used as the control and named as nHAP/ OSA/GT. The columnar gels were placed on the culture dishes coated with S. aureus, which were cultured at 37 °C for 24 h for the detection of inhibition zone. Meanwhile, the columnar gels were soaked in 5 mL SBF, respectively. After 1, 3, 5 and 7 days of soaking, the supernatants were collected and mixed with S. aureus bacteria culture medium respectively in the proportion of 1:1 and coated on the culture dishes with 6 replicates per group. After 24 h of culture, the colony forming units (CFU) of S. aureus were counted by the enumeration method of colonies on plate to evaluate the sustained antibacterial effect of the composites.
a SA
Transmittance (%)
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1417.1 1614.6
OSA 3428.4
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Fig. 3. FTIR spectra of SA, OSA, GT and OSA/GT (a), the hydroxylamine hydrochloride–potential titration curve (b) and the first differential curve (c) of OSA.
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3. Results and discussion 3.1. Characterization of nHAP The implant-related infection is one of the most common complications in arthroplasty. The routine method to prevent the deep infection is using antibiotic-impregnated cement. But the high dose and irrational release cycle of drugs may lead to many undesirable side effects. The ideal strategy is to introduce directly a drug delivery system in the local site of surgery, improving the anti-infection properties for a long time. Hence, self-made nanoparticles had been used as carriers for the treatment of infection. First, chemical composition of the nanoparticles has been characterized using XRD and the predominant HAP crystal phase had been confirmed according to the standard card of HAP (JCPDS 09-432) (as shown in Fig. 1a). The peaks in the pattern were sharp, which implied that the nanoparticles had higher crystallinity.
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The morphology of nHAP was shown in Fig. 1b, in which the particles had an irregular shape and their sizes were ca. 30–40 nm. Some of the particles adhered to each other because of the fusion of crystal during the sintering process. According to the BET method, the specific surface area, pore diameter and porosity of nHAP were analyzed and results were 67.82 m2 g−1, 11.72 nm and 51.74%, respectively. Usually, drug adsorption happened mainly on the surface of adsorbent. So the nHAP particles with bigger specific surface area and higher porosity were conducive to the drug loading in subsequent experiments. 3.2. Load and release of VAN In order to determine the drug loading-release property and bactericidal property of the nHAP particles, VAN has been used as a model drug. The drug loading process has been performed by soaking the nHAP particles in SBF solution containing VAN. The load capacity of
Fig. 4. The rheogram of four OSA/GT samples: a: viscosity–time curves; b: G', G″–time curves.
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a1
b2
b1
a2
Fig. 5. FE-SEM images of OSA/GT (a1) and VAN-nHAP/OSA/GT (a2) composites and digital photos of OSA/GT (b1) and VAN-nHAP/OSA/GT (b2) by the method of TA.XT Plus.
VAN on every 100 mg nHAP was measured using the weight difference method on the base of the curve of VAN content and absorbance (Fig. 2a). VAN (955.68 ± 25.44 μg) has been loaded on every 100 mg nHAP particles and load rate was up to 19.11 ± 0.29%. Based on the cumulative release curve in Fig. 2b, the release process of VAN could be divided into two parts according to its release rate. There was a burst release of VAN at the first 24 h, which reached 61% of total amount of loaded VAN on nHAP, which could be ascribed to VAN adsorbed on the surface of nHAP. The release rate reduced gradually after the initial release phase. Released drug in the second process could be attributed to VAN encapsulated in the porous channels. About 72% of VAN loaded on nHAP was released after 375 h and the release was also last with the increase of immersing time, which suggested that nHAP could be served as an effective carrier to modulate the release of VAN with a slower release rate and a longer release time. 3.3. Characterization of OSA/GT and VAN-nHAP/OSA/GT composites When used in the fracture surface of bones, an ideal carrier material for the treatment of bone infection should not only possess a reasonable drug loading-releasing property but also be fixed in a close site to fracture bone so as to maximize the function of the antibiotic. Hence, a sticky matrix composed of OSA and GT had been constructed to bond VNA-loaded nHAP particles to the bone fracture surface. Sodium periodate, as a strong oxidizing agent, can oxidize the adjacent glycol structure of SA into the adjacent dialdehyde group [26]. The aldehyde group of OSA was demonstrated by the result of FTIR (Fig. 3a). SA, which contains a lot of hydroxyl, has strong polarity and can form hydrogen bonds with electron donors (such as –NH2, –OH, C–O–C) which have lone pair electrons. When hydrogen bond is formed in the SA matrix, stretching vibration frequency of –OH will decrease and the bond shape will become wider [27]. In Fig. 3a, there was a strong and wide diffraction peak of ν(O–H) at 3428.4 cm−1 and a stretching vibration diffraction peak of C–O at 1031.6 cm−1 in the spectrum of SA. Compared with the spectrum of SA, a new characteristic diffraction peak appeared in the spectrum of OSA at 1799.6 cm−1, which could be ascribed to the vibration absorption peak of C = O of the aldehyde group. Along with the oxidation process, the hydrogen bonds were weakened and the stretching vibration frequency of –OH moved from 3428.4 cm−1 to 3446.2 cm−1 and the bond was narrower, which suggested that the aldehyde group of OSA was obtained during the periodate oxidation of SA. According to the hydroxylamine hydrochloride–potential titration curve (Fig. 3b) and its first differential curve (Fig. 3c) of OSA, the aldehyde group concentration ([CHO], mol/g) and degree of oxidation (OD) were calculated, which were 5.491 ± 0.008 mmol/g and 54.39 ± 0.08% respectively. Schiff base can be formed by the reaction of the aldehyde group of OSA and amino of GT. The FTIR spectra of OSA, GT and OSA/GT (Fig. 3a) showed that, the characteristic diffraction peaks of aldehyde group of OSA at 1799.6 cm−1 and amide related to GT at 1653.4 and 1551.3 cm− 1 were weakened in the OSA/GT composite due to the formation of the Schiff base. Besides, there was a strong characteristic
diffraction peak of C=N bond at 1643.0 cm−1, which could be assigned to the characteristic absorption peak of Schiff base [28], and the peak became wider because of the overlapping with the absorption peak at 1653.4 cm−1 of GT. All results showed that Schiff base was obtained due to the crosslinking of OSA and GT. In order to understand their gel transition times, the rheological behaviors of four OSA/GT composites with different ratio of OSA and GT were measured (Fig. 4). The dynamic storage modulus (G') reflects the elasticity of the system, which will overlap with the dynamic loss modulus (G") when the crosslinking system is close to the gel point. So the time of G'=G" can be defined as gel time [29]. In this research, the concentration of OSA and GT selected was 10% and 15% respectively. The viscosity–time curves of OSA/GT with different volume ratios (Fig. 4a) showed that, the viscosity of the system increased with the increase of time. Meanwhile, the viscosity of composite increased with the increase of the volume of GT. The G', G"–time curves of the OSA/GT systems (Fig. 4b) showed the gel times of these composites were 2160 s, 822 s, 330 s and 268 s when the ratios of OSA to GT were 2:1, 1:2, 1:3 and 1:4 respectively, which indirectly implied the change trend of their viscosity over time. Considering the rheological results and operation convenience in practical application, the volume ratio of 10% OSA and 15% GT selected for antibacterial experiment was 1:2. Furthermore, the surfaces of glass plates were coated with both OSA/GT and VAN-nHAP/OSA/GT composites respectively to observe the distribution of VAN-nHAP particles in the composite using FE-SEM (Fig. 5a1–a2). VAN-nHAP particles could be dispersed homogeneously in the composite, which was beneficial to stable and homogeneous release of VAN from the composite. Both composites of OSA/GT and VAN-nHAP/OSA/GT had been measured by the texture analyzer. Unlike the translucence of OSA/GT, VAN-nHAP/OSA/GT composite was ivory due to the presence of VAN-nHAP nanoparticles according to the photographs of composites (Fig. 5b1–b2). Moreover, both composites showed an obvious adhesive ability between operation panel and probe. The specific data of hardness, stickiness and stringiness were obtained and results presented here revealed better reproducibility among replicates in Table 1. Both OSA/GT and VAN-nHAP/OSA/GT composites had certain and similar stickiness. Obviously, the presence of VAN-nHAP particles in OSA/GT composite had not changed evidently gel's stickiness. By the stickiness of OSA/GT composite, VAN-nHAP particles could be fixed directly on the fracture surface of bone. As shown in Fig. 6a, compared with nHAP/OSA/GT, the VAN-nHAP/ OSA/GT composite had an obvious bacteriostatic ring, indicating that the VAN-nHAP/OSA/GT composite has a certain bacteriostatic effect. Fig. 6b showed the sustained antibacterial effect of the composites. It can be seen that there is no significant difference between the colony Table 1 The result of TA.XT Plus of composites. Sample
Hardness (g)
Stickiness (g)
Stringiness (mm)
OSA/GT VAN-nHAP/OSA/GT
13.885 ± 0.002 14.208 ± 0.003
4.411 ± 0.005 5.591 ± 0.003
7.153 ± 0.010 7.159 ± 0.008
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Fig. 6. Results of inhibition zone test and colony count of samples (S. aureus). ⁎⁎p b 0.01.
count of blank control and nHAP/OSA/GT composites, which indicated that samples in the absence of VAN had little bacteriostatic effect. The introduction of VAN into the composites brought a significant decrease (p b 0.01) of the colony count, which implied that VAN-nHAP/OSA/GT composite had an excellent antibacterial effect. With the increase of culturing time, the colony count of blank control and nHAP/OSA/GT composite had no obvious change. However, the colony count of VAN-nHAP/ OSA/GT composite continually decreased from (10.3 ± 0.2) × 103 to (9.96 ± 0.08) × 103, (9.5 ± 0.2) × 103 and (9.1 ± 0.1) × 103 cfu mL−1 with the increase of the culture time from 1 to 3, 5 and 7days respectively. The results demonstrated that the VAN-nHAP/OSA/GT composite had a continuous enhancement of antibiotic resistance. This was mainly attributed to the slow-release of VAN from the nHAP particles, which was consistent with the result of Fig. 2b. 4. Conclusion VAN-loaded HAP nanoparticles have been fabricated and the nanoparticles showed a higher drug loading efficiency and better drug controlled release property. After VAN-loaded nHAP combined with OSA/GT gel, viscous VAN-nHAP/OSA/GT composite was obtained. The composite possessed an adhesive property derived from the gel of OSA and GT, and an obvious long-term and continuous antimicrobial performance derived from VAN-loaded nHAP particles. The sticky and antimicrobial composite may has a potential application in arthroplasty to overcome deep infection and improve the success rate of surgery in a simple and direct manner. Acknowledgement The work is financially supported by the Program for National Natural Science Foundation of China (51372226, 51172207, 51202219) and Qianjiang Talent Project of Zhejiang Province (2013R10062).
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