Conferring materials with antibacterial properties 3

CHAPTER NOVEL STRATEGIES FOR CONFERRING ANTIBACTERIAL PROPERTIES TO BONE CEMENT 115 Chapter 6.1 Antibacterial and Mechanical Properties of Bone Cement Impregnated with Chitosan Nanoparticles 6.1.1 Introduction The use of poly(methyl methacrylate) (PMMA)-based bone cement for the anchoring of artificial joints started in 1958 when Sir John Charnley first succeeded in anchoring femoral head prostheses in the femur using auto-curing PMMA (Kühn, 2000). The PMMA cements in use today bear many similarities with the original cement used by Charnley (Daniels et al., 1998). Commercial bone cements are supplied as two component systems consisting of a powder and a liquid that are mixed in the operating room during the surgical procedure and delivered to the implant site. The powdered portion of the cement contains PMMA particles (~ 10 to 150 μm in diameter), an initiator for polymerization (benzoyl peroxide) and a radiopaque medium (such as barium sulfate or zirconium dioxide). The main ingredient of the liquid portion is MMA monomer. The other ingredient, N,N-dimethyl-p-toluidine (DMpT), acts as an activator in the polymerization reaction and the liquid is stabilized with a small amount of hydroquinone to prevent polymerization during storage of the product and provide a sufficient shelf life. When the solid and liquid components of the bone cement system are mixed, the BPO from the powder and the DMpT from the liquid participate in a redox reaction that produces free radicals which initiate addition polymerization of the MMA monomer. As the polymerization reaction proceeds, the powder/liquid mixture undergoes a transition from the liquid state to a dough and finally to the solid state, forming a rigid, amorphous polymer. The infection rates in primary joint replacement range from 1% to 3% despite strict antiseptic operative procedures, including systemic antibiotic prophylaxis and special enclosures using laminar flow (Hendriks et al., 2004; Harris and Sledge, 1990). Failure 116 Chapter to treat and eradicate biomaterial-associated infection with systemic antibiotic regimens is usually due to the fact that implant infection is associated with biofilm formation. Implant-associated infections in orthopedic surgery are thought to be mainly due to S. aureus, S. epidermidis and Pseudomonas or other Gram-negative rods (Van de Belt, 2001). Such infections can rarely be eradicated without removal or revision of the infected implant. It is expected that as the population ages, more patients will require joint replacement surgery, and when such orthopaedic prostheses are complicated by infection, management is usually highly problematic, and significant morbidity and increased costs are expected (Segreti, 2000). Antibiotic-loaded bone cements (ALBC) have been in use for over 30 years and have been the standard of care in arthroplasty. The clinical benefits of ALBC combined with systemic antibiotic prophylaxis have been reported in a study with more than 22,000 patients (Engesæter et al., 2003). However, a number of problems related to their use still persist. It is known that the antibotics are released from the ALBC in a biphasic fashion, with an initial peak release followed by slow release which may continue for days to months (Hendriks et al., 2004). Hence, while the ALBC may be effective in preventing bacterial infection in the initial period, this protective effect is generally lost if bacterial contact with the cement occurs after a delay of several weeks. Furthermore, there is a worry that the long-term low concentrations of antibiotic around the implant site may well lead to the occurrence of antibiotic-resistant strains, which may then require the addition of a second antibiotic to the bone cement (Neut et al., 2005). An increase in the antibiotic content may extend the period of protection against biofilm formation but aminoglycosides including gentamicin have considerable nephrotoxic side-effects (Parlakpinar et al., 2006) and the mechanical strength of the cement may 117 Chapter also be compromised since high quantities of antibiotics may lead to incomplete polymerization of the cement (Neut et al., 2005). As described in Section 2.2, a number of surface functionalization techniques have been developed to confer substrates with antibacterial properties. However, the use of such techniques is confined to preformed substrates and is not suitable for bone cement since the cement is mixed into a doughy mass just prior to insertion into a prepared cavity. Previous works on conferring antibacterial properties to bone cement have relied on loading with antibiotics (Van de Belt et al., 2001) or silver (Dueland et al., 1982; Vik et al., 1985). However, the increasing use of ALBC may lead to the development of resistant strains of bacteria, and the antibiotic action is also relatively short-lived. Although silver has a broad antibacterial effect, high concentrations of silver ions may have cytotoxic effects (Dueland et al., 1982; Vik et al., 1985), and the use of nanosized silver in bone cement has been proposed to be a better alternative (Alt et al., 2004). In our present work, we explored a strategy based on the incorporation of chitosan (CS) into bone cement. CS (poly(1,4),-β-D-glucopyranosamine), an abundant natural biopolymer (structure shown below), is derived by the deacetylation of chitin obtained from the shells of crustaceans and can be fabricated into film, fiber, bead, and powder forms. HO O HO O O HO NH2 O HO NH x y O Chemical formula of of chitosan 118 Chapter It has great potential as a biomaterial due to its biological activities, low toxicity toward mammalian cells (Kumar et al., 2004; Sashiwa and Aiba, 2004) and antibacterial activity in controlling growth of bacteria and inhibiting viral multiplication (Huh et al., 2001; No et al., 2002; Rabea et al., 2003). To increase the antibacterial activity and solubility of CS, quaternary ammonium CS derivatives (QCS) have been prepared and their antibacterial activities were shown to increase with increasing chain length of the alkyl substituent (Kim et al., 1997). However, one concern is that the incorporation of CS into bone cement may be expected to result in degradation of the mechanical properties of the bone cement. To address this concern, CS and QCS in nanoparticulate form (NP) were used in our modification of two types of commercial bone cement. Mechanical testing was performed, and the antibacterial activities of the modified bone cements were assessed against Gram-positive S. aureus and S. epidermidis. These two bacteria were chosen since they are commonly associated with infections of orthopaedic implants, wounds and indwelling medical devices (Gristina, 1987; Schierholz and Beuth, 2001). 119 Chapter 6.1.2 Experimental Materials and reagents Two types of bone cement with and without gentamicin were used: CMW Smartset and CMW Smartset-G (DePuy International Ltd., UK), and Palacos R and Palacos R-G (Biomet, Merck, Germany). The compositions of the bone cements, as given in the manufacturers’ information leaflets, are shown in Table 6.1. CS was purchased from CarboMer Inc and refined twice by dissolving it in dilute acetic acid (HOAc) solution. The solution was filtered and the CS was precipitated with aqueous sodium hydroxide and then dried in a vacuum oven for 24 h at 40 oC. The viscosity-average molecular weight was about 2.2×105 as determined by the viscometric method (Zhang et al., 2004). The degree of deacetylation was 84% as determined by elemental analysis using the Perkin-Elmer Model 2400 elemental analyzer (Zhang et al., 2004). Table 6.1 Composition of the bone cements used a Smartset Palacos R Smartset G Palacos R-G (w/w%) (w/w%) (w/w%) (w/w%) 84.00 83.88 80.46 82.15 15.00 1.00 15.32 0.80 14.37 0.96 4.22 15.01 0.78 2.06 97.50 2.50 97.98 2.02 97.50 2.50 97.98 2.02 Powder Methylmethacrylate methacrylate copolymer Zirconium dioxide Benzoyl peroxide Gentamicin sulfate Liquid Methylmethacrylate N,N-dimethyl-p-toludine a % by weight (w/w) of powder component and liquid component, respectively. Chlorophyll and hydroquinone are not mentioned in this table. S. aureus (ATCC 25923) and S. epidermidis (ATCC 12228) were obtained from American Type Culture Collection. The minimum inhibitory concentration (MIC) of gentamicin as determined by the broth dilution method recommended by the National 120 Chapter Committee for Clinical Laboratory Standards (2003) is 0.12 μg/ml for S. aureus and 0.025 μg/ml for S. epidermidis. Synthesis of QCS QCS was synthesized according to the method previously reported (Huh et al., 2001). In brief, g of CS was added to 50 ml N-methyl-2-pyrrolidinone and suspended by stirring at room temperature for 12 h. The temperature of the suspension solution was then lowered to oC using an ice water bath. A 1.5 N NaOH aqueous solution (15 ml), potassium iodide (1.2 g) and hexylbromide (13 g) were added to this solution and its temperature was raised to 45 oC and maintained at this value for 48 h while stirring. The reaction solution was then filtered using a mesh (120 mesh) to remove the insoluble portion. The filtrate was precipitated into a large excess of acetone and filtered using a filter paper. The precipitate was re-dispersed and washed with acetone times and the resulting product was dried under vacuum. Preparation of CS NP and QCS NP CS NP was prepared using the ionic gelation method (Calvo et al., 1997). CS was dissolved in v/v% HOAc solution at a concentration of 0.5 w/v% and the pH was raised to 4.6-5 with 10 N NaOH. CS NP was formed upon adding ml of 0.25% TPP in water to 15 ml CS solution under stirring at a speed of 1000 rpm. The nanoparticles were separated by centrifugation at 20000 rpm for 30 min. The supernatant was discarded and the CS NP was extensively rinsed with water to remove any NaOH and then freeze-dried before further use. QCS NP was obtained from QCS using the same method. 121 Chapter Characterization The chemical composition of the surfaces was analyzed by XPS as described in Section 3.1.2. The nanoparticles were examined using a FE-SEM (JEOL JSM 6700F) and the size and size distribution were determined by laser light scattering with a Brookhaven LLS 90 Plus Particle Size Analyzer. The dried nanoparticles were first suspended in water and sonicated to obtained a homogeneous suspension before the measurement. The zeta potential of the nanoparticles was measured by a zeta potential analyzer (Zeta Plus from Brookhaven Instruments) with palladium electrodes, and the mean of six readings was calculated. Preparation of cements Chitosan in the form of powder or nanoparticles was mixed with bone cement powder at weight ratios of 15:100 and 30:100. All the cements were prepared by manually mixing the powder with the liquid monomer in a ratio of g/ml in a bowl in a laminar flow hood, in accordance with the manufacturer's instructions. The polymer powder was placed in a bowl and the monomer was added and stirred using a spatula until the powder was fully wetted. The mixture was subsequently inserted into the mould at approximately dough time, usually about min. The filled mould was pressed between two glass plates for h. After the cement had hardened, it was pulled out of the mould and stored under dark, sterile conditions at room temperature. Different moulds were used to make samples for the different tests. Rectangular beams (25 × 10 × mm3) were used for the bending tests, while the specimens for the tensile tests were 75 mm in length, mm in width, mm in thickness, with a gauge length of 25 mm (Harper and Bonfield, 2000). Cylindrical specimens (6 mm in diameter and mm in height) and rectangular specimens were prepared for the cytotoxicity and antibacterial assays 122 Chapter respectively. The preparation of bone cement with the QCS nanoparticles was carried out in a similar manner. Testing of mechanical properties The tensile test and three-point bending test were performed on the Instron universal materials testing machine (Model 5544). The tensile test (according to ASTM D638-03) was conducted at a cross-head speed of mm/min. For the three-point bending test (according to ASTM D790-3) the span length was 20 mm and the loading rate was mm/min. Five specimens were used in each mechanical test. The bending modulus (EB) was calculated according to Eq. (1): EB = L3m/4bd3 (1) where L is support span (mm), b is width of beam tested (mm), d is depth of beam tested (mm), and m is slope of the tangent to the initial straight-line portion of the load-deflection curve (N/mm). Determination of antibacterial activity Two Gram-positive bacterial strains S. aureus and S. epidermidis were cultivated as described in Section 3.1.2. An aliquot (2 ml) of culture was then added to the yeast-dextrose broth and incubated for 6-8 h at 37 oC until the exponential growth phase was reached. 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(30 %), the other substrates were mixed at a weight ratio of CS or QCS to PMMA bone cement powder of 15% (#), (*) denote significant differences (P < 0.05) compared with original bone cement which is freshly prepared or after 3 weeks in PBS, respectively 130 Chapter 6 4.0 (a) Freshly prepared o After 3 weeks in PBS at 37 C Bending modulus (GPa) 3. 5 3. 0 # * # * 2.5 2.0 1.5 1.0 0.5 0.0 Original CS (30 %)... Freshly prepared o After 3 weeks in PBS at 37 C # 6 # * 2 Log(CFU/cm ) 5 4 # * 3 2 1 0 Original 7 CS (b) QCS NP Freshly Prepared o After 3 weeks in PBS at 37 C 6 # * 5 # * 2 Log(CFU/cm ) CS NP 4 3 # * 2 1 0 Original CS CS NP QCS NP Figure 6.6 Number of viable adherent S aureus (a) and S epidermidis (b) cells on the different substrates based on Smartset bone cement without gentamicin 133 Chapter 6 The QCS... prepared o After 3 weeks in PBS at 37 C (a) Young's modulus (GPa) 2.5 * 2.0 # * 1.5 1.0 0.5 0.0 Original CS 30 % CS CS NP QCS NP 3. 0 Freshly prepared o After 3 weeks in PBS at 37 C (b) Young's modulus (GPa) 2.5 2.0 * 1.5 1.0 0.5 0.0 Original CS CS NP QCS NP Figure 6.4 Young’s modulus of original and CS (30 %), CS, CS NP and QCS NP-loaded Smartset plain (a) and gentamicin-loaded (b) bone cements With the exception... suspension (108 S aureus cells/ml, for 3 h at 37 oC) 138 Chapter 6 Effect of different bone cements The antibacterial assay results discussed so far in Section 6.1 .3 have been obtained with the Smartset and Smartset-G bone cements Similar tests were carried out with the Palacos R and Palacos R-G bone cements and the results are summarized in Table 6 .3 Table 6 .3 Number of viable adherent S aureus and S... * 2.5 2.0 1.5 1.0 0.5 0.0 Original CS (30 %) 3. 5 (b) CS CS NP QCS NP Freshly prepared o After 3 weeks in PBS at 37 C Bending Modulus (GPa) 3. 0 * 2.5 2.0 1.5 1.0 0.5 0.0 Original CS CS NP QCS NP Figure 6.5 Bending modulus of original and CS (30 %), CS, CS NP and QCS NP-loaded Smartset plain (a) and gentamicin-loaded (b) bone cements With the exception of CS (30 %), the other substrates were mixed at a weight... lower MIC for S epidermidis than S aureus However, after 3 weeks immersion in the PBS, its effectiveness against this bacterium is also greatly reduced It has been reported that 134 Chapter 6 1000 Freshly prepared o After 3 weeks in PBS at 37 C (a) 800 * CFU/cm 2 600 400 * 200 * # # 0 Original CS 600 2 QCS NP Freshly prepared o After 3 weeks in PBS at 37 C (b) CFU /cm CS NP 400 * 200 * 0 Original CS CS... CS or QCS to PMMA bone cement powder of 15% 131 Chapter 6 trend (Figure 6.4(b) and Figure 6.5(b)) In view of these results, the antibacterial assays were carried out with composites having a CS or QCS to bone cement powder weight ratio of 15% in order to achieve a balance between mechanical strength and antibacterial effectiveness Antibacterial assay The antibacterial effects of the various bone cements... obtained with these bone cements are not significantly different from that of the non-toxic control (growth culture medium) Table 6.4 Cytotoxicity assay of 3T3 cells on the different bone cements Substrates Growth medium PMMA bone cement * CS-loaded bone cement * CS NP-loaded bone cement * QCS NP-loaded bone cement Triton X-100 (1 wt%) * Cell viability % of control 100 ± 2 98 ± 4 1 03 ± 3 97 ± 3 98 ±... interaction with the cell membrane of bacteria, which is usually negatively charged As shown in Table 6.2, the zeta potential increases from 48 mV to 67 mV after quaternization Table 6.2 Characteristics of CS NP and QCS NP Nanoparticles CS QCS Mean diameter (nm) 220 ± 3 284 ± 2 Polydispersity 0.16 ± 0.02 0. 13 ± 0.01 Zeta potential (mV) 48.87 ± 0.46 67 .32 ± 0.14 127 Chapter 6 (a) (b) (c) Figure 6 .3 FE-SEM... cement are given in Figure 6.7 It can be seen that some S aureus bacteria can remain viable after contact with the gentamicin-loaded cement for 3 h (Figure 6.7(a)), which is probably due to the high bacterial cell concentration in the nutrient broth After 3 weeks immersion in PBS at 37 °C, the antibacterial effectiveness of the gentamicin-loaded bone cement decreases significantly and the number of . 6 NOVEL STRATEGIES FOR CONFERRING ANTIBACTERIAL PROPERTIES TO BONE CEMENT Chapter 6 116 6.1 Antibacterial and Mechanical Properties of Bone Cement Impregnated with Chitosan Nanoparticles. 37 o C (b) * Chapter 6 131 Original CS (30 %) CS CS NP QCS NP 0.0 0.5 1.0 1.5 2.0 2.5 3. 0 3. 5 4.0 Bending modulus (GPa) Freshly prepared After 3 weeks in PBS at 37 o C (a) # * # * Original. cements was investigated by staining with a combination dye as described in Section 5.2. In vitro cytotoxicity assay 3T3 mouse fibroblasts cells (3T3-Swiss albino, ATCC) culture and cell