Towards Elimination of Pin-tract Infections: Novel Antibacterial Coating on Orthopaedic Wires

35 Pages·2017·9.34 MB·English

AAC Accepted Manuscript Posted Online 8 May 2017
Antimicrob. Agents Chemother. doi:10.1128/AAC.00442-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
1 Towards Elimination of Pin-tract Infections: Novel Antibacterial Coating on Orthopaedic Wires
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4 Dmitry Gil,a Sergey Shuvaev,b Anastasia Frank-Kamenetskii,a Vladimir Reukov,a,c Christopher
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5 Gross,d Alexey Vertegela# o
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7 Department of Bioengineering, Clemson University, Clemson, SC, USAa; Department of e
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8 Chemistry, Durham University, Durham, UKb; Institute for Biological Interfaces of Engineering, ro
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9 Clemson University, Clemson, SC, USAc; Department of Orthopaedics, Medical University of h
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10 South Carolina, Charleston, SC, USAd //
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11 .
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12 Running head: Monolaurin Coating on Orthopaedic Implants .
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13 /

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14 #Address correspondence to Alexey Vertegel, [email protected] N
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16 Abstract:
17 Novel approaches to prevention of microbial infections after insertion of orthopedic external
18 fixators are in great demand because of extremely high incidence rate of such infections, which
19 can reach up to 100% for longer implant residence times. Monolaurin is an antimicrobial agent
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20 with known safety record, broadly used in food and cosmetic industries; however, its use in o
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21 antimicrobial coatings of medical devices has not been studied in much detail. Here, we report lo
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22 the use of monolaurin as an antibacterial coating on external fixators for the first time. The e
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23 monolaurin-coated Kirschner wires (K-wires) showed excellent antibacterial properties against o
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24 three different bacterial strains – methicillin-sensitive Staphylococcus aureus (MSSA),
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25 methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis. A ca. //
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26 6.0-log reduction of both planktonic and adherent bacteria was achieved, when using .
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27 monolaurin-coated K-wires. At the same time, monolaurin-coated K-wires did not show any .
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28 observable cytotoxicity with mouse osteoblast cell cultures. Overall, monolaurin-coated K-wires /

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29 could be promising as potent antimicrobial materials for orthopaedic surgery. N
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30 e
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31 1. Introduction e
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32 Kirschner wires (K-wires) are extensively used in orthopaedic applications ranging from 6
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33 fracture fixation to deformity correction. Despite being very versatile and fairly effective, the K- 1
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34 wiring procedure is often associated with common pin-tract infections that can lead to even more y

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35 serious issues such as osteomyelitis (1, 2). These infections arise from the use of percutaneous e
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36 pinning techniques, as seen in skeletal traction, percutaneous fracture pinning, and external
37 fixation for fracture stabilization or complex deformity reconstruction. Pin sites are niduses for
38 infections since the skin barrier is disrupted, providing a potential passage for opportunistic
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39 bacteria to enter a previously privileged area. Multiple evidence suggests that the infection rate
40 following a K-wiring procedure ranges from 11 to 100 %, depending on the lifetime of the
41 implant (3–5). If appropriate pin care is neglected, these infections can cause sepsis,
42 osteomyelitis and sometimes mortality (3, 6). The economic burden of treatment for this this type
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43 of infections is expected to reach as high as $1.6B annually by 2020 (7). Therefore, surgeons, o
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44 microbiologists, and material scientists alike are scrambling to find the best possible solutions to lo
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45 this challenging issue. e
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46 In attempts to prevent pin track sepsis, multiple strategies have been developed. Most of them o
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47 involve coating the implants with antibiotics, antimicrobial or anti-adhesion polymers and
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48 peptides; silver or nitric ions; nanoparticles; and other antiseptics like chlorhexidine and silver- //
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49 sulfadiazine (8–12). However, a major concern associated with the implementation of .
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50 antibacterial coatings is their adverse effect on adjacent tissues and bones, which inhibits fracture .
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51 or fusion healing (13). Another important issue is the in vivo loss of antibacterial activity /

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52 resulting from the exposure of bacteria to sub-lethal concentrations of the drug, eventually N
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53 leading to development of drug resistance in situ (14). Hence, the development of novel coatings e
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54 for reducing pin-site infections is of particular importance. e
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55 Antibacterial properties of fatty acids and their esters have been extensively studied over the 6
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56 past few decades. Monolaurin, also known as glycerol monolaurate, was found to be the most 1
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57 effective antibacterial agent among these compounds (15). Monolaurin is currently used as a y

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58 broad range antimicrobial agent in food and cosmetic industries. Multiple studies have shown e
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59 that besides antibacterial properties monolaurin also possesses antiviral and antifungal activity
60 (16–18). In an in vitro studies, monolaurin prevented biofilm formation of various
61 Staphylococcus species including Staphylococcus aureus (S. aureus) and Staphylococcus
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62 epidermidis (S. epidermidis), pathogens that cause approximately 80% of orthopaedic implant-
63 associated infections [18,19, 20]. It was also shown that at sub-lethal concentrations, bacteria did
64 not develop resistance to monolaurin (21). In vivo experiments demonstrated that oral
65 administration of monolaurin appeared to be an effective approach to manage and treat S. aureus
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66 infections (19). In a surgical site infection model (22), monolaurin exhibited antibacterial and o
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67 antibiofilm activity against both gram-positive and gram-negative bacteria. Therefore, a large lo
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68 body of evidence suggests that monolaurin possesses profound antimicrobial properties, which e
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69 make it an excellent candidate for antibacterial coating on orthopaedic devices, in particular K- o
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70 wires and external fixation devices, because of high incidence rates of infections for these
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71 devices. Advantages of monolaurin as a coating include its low cost, ease of application and //
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72 “Generally Recognized as Safe” (GRAS) status by the FDA; in addition, monolaurin’s low water .
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73 solubility may provide for a prolonged drug release rate from the implant and improved .
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74 antimicrobial performance. /

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75 In the study reported here, monolaurin is employed as a passive coating on K-wires with the N
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76 long-term goal of achieving reduced incidence rate of orthopaedic implant-associated infections e
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77 and decrease the burden of pin tract infections, thus improving patient outcomes. A dip-coating e
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78 technique was implemented to modify stainless steel K-wires. Following physicochemical 6
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79 characterization of the coating, the antibacterial properties of modified wires were evaluated in 1
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80 vitro in the model experiments with S. epidermidis, methicillin-sensitive S. aureus (MSSA) and y

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81 methicillin-resistant S. aureus (MRSA). Furthermore, the biological effect of monolaurin coating e
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82 on osteoblasts was studied. The objective of this study was to demonstrate the efficacy of
83 monolaurin-coated K-wires in vitro and provide a foundation for further animal and clinical
84 studies.
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86 2. Results
87 2.1. Binding yield of monolaurin. Binding yield of monolaurin was determined based on the
88 adsorption isotherm of the drug on stainless steel K-wires (Fig. 1). Due to the low molar
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89 extinction coefficient of monolaurin, a large error was observed at low loading concentrations. o
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90 The adsorption isotherm reached saturation at monolaurin concentration of approximately 7.5 lo
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91 mg/ml. The saturation loading was found determined to be 84±4 μg of monolaurin per 1 cm e
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92 wire, corresponding to the density of 4.3±0.2 μg/mm2. Based on these results, 10 mg/ml o
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93 monolaurin solutions were used to coat K-wires in all future experiments to ensure saturation t
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94 coating. /a
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95 2.2. Characterization of monolaurin coating. Fourier transform infrared spectroscopy .a
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96 (FTIR) was used to prove monolaurin adsorption on the K-wires. Fig. 2 shows the spectra .
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97 collected from coated and pure monolaurin. /
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98 As can be seen, there is virtually no difference between the spectra of the coated sample and N
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99 that of the pure monolaurin. At the same time, the spectrum of a plain wire (not presented here) m
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100 does not display any absorbance bands. Thus, these data confirm that the coating consists of r

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101 monolaurin. ,

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102 Morphological characteristics of the coating were analyzed using scanning electron 1
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103 microscopy (SEM), atomic force microscopy (AFM) and spectroscopic reflectometry. As shown y

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104 in Fig. 3b, monolaurin-coated wires exhibit smooth surface with minor extrusion lines. e
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105 According to the micrographs, the coating is evenly distributed across the surface of the wire.
106 Little to no difference can be seen when comparing surface morphology of the plain and
107 monolaurin-coated K wires.
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108 Further analysis of micromorphological characteristics of the monolaurin coating was
109 conducted using AFM technique. Topography images acquired by AFM (Fig. 4a-d) confirmed
110 the smoothness of monolaurin coating. Using scratching technique, the thickness of the coating
111 was determined to be 144±35 nm. This results are in good correlation with those of the
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112 spectroscopic reflectometry, which showed the thickness of the coating to be 161±57 nm. Coated o
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113 samples exhibited considerably higher adhesion force between the surface and the cantilever tip lo
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114 compared to the uncoated samples (85.2±18.6 and 15.9±2.1 nN for coated and uncoated wires, d

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115 respectively, p<0.001), indicating the presence of a uniform coating (Fig. 4e-f). m

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116 2.3. Evaluation of antibacterial activity. t
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117 2.3.1. Antibacterial activity against planktonic bacteria. The antimicrobial activity of /a
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118 monolaurin-coated wires against planktonic bacteria was assessed based on the microbial .a
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119 viability curves. As shown in Figs. 5, S3, coated wires were highly efficient against MSSA, .
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120 MRSA and S. epidermidis, completely eliminating planktonic bacteria in less than 7.5 hours. In /
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121 the presence of the uncoated wires, the number of bacteria increased by more than two orders of N
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122 magnitude over the first 7.5 hours. These data suggested that monolaurin-coated wires achieved m
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123 a 6.0-log reduction of planktonic MSSA, MRSA and S. epidermidis. r

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124 2.3.2. Antibacterial activity against adherent bacteria. The antibiofilm activity of monolaurin ,

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125 coating was evaluated by the Crystal Violet (CV) assay and quantitative analysis of adherent 1
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126 bacteria. The results of the CV assay are shown in Fig. 6a. In the case of all three bacterial y

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127 strains, the absorbance values for coated wires were significantly lower when compared to those e
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128 for the plain wires, indicative of antibiofilm activity of monolaurin-coated wires against MSSA,
129 MRSA and S. epidermidis (p<<0.05, n=6).
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130 Figs. 6b, S3 show the count of adherent bacteria. Again, monolaurin-coated wires
131 demonstrated pronounced antibiofilm properties resulting in the significant drop of the number
132 of adherent bacteria when compared to the plain wires (p<<0.05, n=4). Monolaurin-coated wires
133 effected a 5.7-, 5.8- and 6.0-log reduction of adherent MSSA, MRSA and S. epidermidis,
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134 respectively. These results are consistent with those obtained for planktonic bacteria. o
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135 The observed antibiofilm effect was additionally confirmed by SEM imaging. As can be seen lo
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136 from Fig. 7 little or no evidence of adherent bacteria is observed on the surface of the coated e
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137 wires for all three bacterial strains studied herein. On the other hand, a large number of adherent o
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138 bacteria surrounded by extracellular matrix are evident on the SEM images of the uncoated wires
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139 for all three strains studied. The analysis of high magnification images (30,000X) revealed that //
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140 the morphology of bacteria adherent to the plain wires is different when compared to those found .
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141 on monolaurin-coated wires (Figs. 8, S4, S5). It is evident that in the latter case, the integrity of .
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142 bacterial cell wall is disrupted, resulting in the deformation of the bacterial cell surface. In /

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143 contrast, the bacteria attached to the plain wire exhibit the conventional spherical shape. N
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144 2.3.3. Inhibition zone test. Additional evaluation of antimicrobial activity of monolaurin-coated e
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145 wires was performed by the inhibition zone assay. As shown in Fig. 9d, coated wires were e
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146 efficient against all three bacterial strains tested. The results of time-dependent antimicrobial 6
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147 efficacy test shown in Figs. 9a-c suggest that the inhibition diameter decreases significantly with 1
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148 time (p<0.05, n=6). However, even after 96 hours of incubation, monolaurin-coated K-wires y

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149 were capable of inhibiting bacterial growth for MSSA, MRSA and S. epidermidis. e
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150 Overall, results of the inhibition zone test are indicative of good prolonged antibacterial
151 efficacy of monolaurin-coated wires, demonstrating their ability to inhibit growth for all three
152 strains for at least 4 days.
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153 2.4. Evaluation of cytotoxicity. The influence of monolaurin coating on cell proliferation was
154 assessed using the methyl thiazolyl tetrazolium (MTT) assay (Fig. 10a). The image demonstrates
155 that culturing osteoblasts in the presence of monolaurin-coated wires did not affect cell
156 proliferation rate: the OD , which indirectly corresponds to the number of cells, was not
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157 significantly different for coated and uncoated wires at different time points (p=0.687, 0.781, o
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158 0.757, for day 2, 4, and 7, respectively; n=4). lo
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159 The analysis of the adherent cells was performed using SEM imaging and the Live/Dead e
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160 assay. Figs. 10c-f display micrographs of the osteoblasts adherent to the surface of plain and o
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161 monolaurin-coated wires. Good cell attachment and spread across the surface is evident from t
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162 these images; osteoblasts exhibited normal polygonal morphology with extended filopodia. Little /a
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163 to no difference can be noticed when comparing plain and modified samples. .a
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164 Fig. 10b shows fluorescent images of osteoblasts stained with Live/Dead reagents. Similarly .
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165 to plain wires, monolaurin-coated wires exhibited a large number of adherent osteoblasts, vast /
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166 majority of which were found to be viable (Table S1). Quantification of obtained results revealed N
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167 that the percentage of viable cells not significantly different for plain (99.4±0.1 %) and m
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168 monolaurin-coated (99.4±0.1 %) wires and tissue-grade polystyrene (99.5±0.1 %), which was r

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169 used as a positive control (p=0.311, n=4). ,

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170 Thus, the results of SEM imaging and the Live/Dead assay are in good agreement with the 1
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171 results of the MTT assay, and suggest good biocompatibility and no cytotoxicity of monolaurin-
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172 coated K-wires. s
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174 3. Discussion
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175 The present work focusses on potential benefits of using monolaurin as an antibacterial coating
176 on orthopaedic K-wires in attempt to reduce the burden of pin-tract infections, in particular those
177 caused by S. aureus (including MRSA) and S. epidermidis.
178 In this study, stainless steel K-wires were coated with monolaurin using a simple yet effective
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179 dip-coating method. The proposed methodology appears to be fairly inexpensive and simple, and o
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180 does not require the use of toxic chemical compounds or thermal/mechanical treatment that may lo
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181 cause structural, chemical or physical alterations in the implants. The “GRAS” status of ethanol e
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182 and monolaurin, the only compounds used for the manufacturing of the coated K-wires, can o
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183 facilitate the translation of modified wires into clinical practice.
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184 Given that binding yield of monolaurin at saturation was found to be ca. 4.3 μg/mm2, the total //
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185 dose of monolaurin absorbed on a 1 cm K-wire is 84 μg. Although the antibacterial activity of .
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186 monolaurin has been studied previously, there are still debates regarding the therapeutic dose and .
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187 the minimally inhibitory concentration (MIC) of this compound. Having analyzed the /

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188 antibacterial activity of monolaurin against 29 strains of S. aureus, including MRSA, K. Holland N
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189 et al (23) determined that its MIC is in the range from 10 to 20 μg/ml. However, H. Preuss et al e
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190 (24) calculated the MIC of monolaurin against S. aureus to be 62.5 μg/ml. Another study e
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191 suggests that the MIC of monolaurin against S. aureus is in the range from 25 to 50 μg/ml (25). 6
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192 At the same time, we were unable to find the MIC of monolaurin against S. epidermidis in the 1
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193 literature. However, the antimicrobial activity of lauric acid against S. epidermidis is well y

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194 studied: the MIC was determined to be 3.9 μg/ml (26). Multiple studies suggest that monolaurin e
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195 has better antimicrobial properties than lauric acid (21, 25, 27). Therefore, the MIC of
196 monolaurin against S. epidermidis can be expected to be lower than 3.9 μg/ml. Overall, even if it
197 is difficult to estimate the volume of liquid to which a K-wire is exposed in vivo after the
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198 implantation, the dose of monolaurin introduced with K-wires in this study (84 μg per one cm of
199 wire) is expected to provide antibacterial properties to the implants at least during the first days
200 after the implantation. Results of our inhibition zone tests support this conclusion. Nonetheless,
201 further animal studies are required to determine if the current dose is sufficient to provide a
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202 therapeutic effect in vivo. o
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203 There is a general consensus that increased surface roughness leads to the enhanced lo
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204 osteointegration, primarily due to the more developed bone-to-implant contact (28, 29). e
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205 However, in certain cases improved osteointegration can be undesirable (30). The use of external o
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206 fixators and K-wires implies the subsequent removal of the implants, and a more developed
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207 bone-to-implant contact would increase frictional forces between the implant and bone upon //
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208 removal, causing severe discomfort for patients. Therefore, alterations of surface characteristics, .
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209 in particular roughness, of external fixators and K-wires should be avoided. The results of SEM .
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210 and AFM imaging conducted herein showed that topography of the monolaurin-coated wires was /

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211 similar to that of the plain wires (Figs. 3, 4). The only parameter that was affected by the N
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212 presence of monolaurin coating is the surface stickiness, as measured by AFM. However, an e
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213 observed increase of stickiness is not expected to affect cell adhesion and osteointegration in any e
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214 way, since those responses are guided biochemically (through adhesion proteins), not physically. 6
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215 Hence, the modification of K-wires with monolaurin coating is not anticipated to influence 1
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216 osteointegrative properties of the implants. One possible issue that can be caused by the altered y

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217 stickiness of the wires is their handling by the surgeon during the implantation and removal. e
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218 Here, we qualitatively tested handling of the coated wires in a pilot experiment by using a wire to
219 pierce a silicon rubber Septa flask cork, and did not see any difference between handling coated
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