Shanghai Key Laboratory of Orthopaedic Implant, Department of Orthopaedic Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University, School of Medicine, China.
Biomaterials. 2012 Jan;33(2):365-77. doi: 10.1016/j.biomaterials.2011.09.084. Epub 2011 Oct 20.
Biomaterial-associated infections remain a serious complication in orthopaedic surgery. Treatments, including the local use of antibiotic-loaded polymethylmethacrylate (PMMA) bone cement, are not always successful because of multiantibiotic-resistant organisms. In this study, we synthesised a new quaternised chitosan derivative (hydroxypropyltrimethyl ammonium chloride chitosan, HACC) that contains a series of substitutions of quaternary ammonium and demonstrated that HACC with a 26% degree of substitution (DS; referred to as 26%HACC) had a strong antibacterial activity and simultaneously good biocompatibility with osteogenic cells. We loaded 26%HACC at 20% by weight into PMMA bone cement to investigate whether HACC in PMMA prevents bacterial biofilm formation on the surface of bone cements. Chitosan-loaded PMMA (at the same weight ratio), gentamicin-loaded PMMA and PMMA with no antibiotic were also investigated and compared. Two clinical isolates, Staphylococcus epidermidis 389 and methicillin-resistant S. epidermidis (MRSE287), and two standard strains, S. epidermidis (ATCC35984) and methicillin-resistant Staphylococcus aureus (ATCC43300), were selected to evaluate the bacterial biofilm formation at 6, 12 and 24 h using the spread plate method, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The results showed that 26%HACC-loaded PMMA inhibited biofilm formation on its surface, while the PMMA control and chitosan-loaded PMMA were unable to inhibit biofilm formation. The gentamicin-loaded PMMA decreased the number of viable methicillin-resistant Staphylococcus strains, but its ability to inhibit biofilm formation was lower than 26%HACC-loaded PMMA. Real-time PCR demonstrated that 26%HACC-loaded PMMA markedly downregulated the expression of icaAD, which encodes essential enzymes for polysaccharide intercellular adhesion (PIA) biosynthesis, upregulated the expression level of icaR, which negatively mediates icaAD expression, and also downregulated the expression of MecA, which encodes membrane-bound enzymes known to be penicillin-binding proteins. Our study indicates that 26%HACC-loaded PMMA prevents biofilm formation of Staphylococcus, including antibiotic-resistant strains, on the surface of bone cement, and downregulates the virulence-associated gene expression of antibiotic-resistant staphylococcus, thus providing a promising new strategy for combating implant infections and osteomyelitis.
生物材料相关感染仍然是骨科手术中的严重并发症。由于多抗生素耐药菌的存在，包括局部使用载抗生素聚甲基丙烯酸甲酯（PMMA）骨水泥在内的治疗方法并不总是成功。在这项研究中，我们合成了一种新的季铵化壳聚糖衍生物（羟丙基三甲基氯化铵壳聚糖，HACC），它包含一系列季铵取代物，并证明具有 26%取代度（DS；称为 26%HACC）的 HACC 具有很强的抗菌活性，同时与成骨细胞具有良好的生物相容性。我们将 26%HACC 以 20%的重量加载到 PMMA 骨水泥中，以研究 HACC 在 PMMA 中是否能防止细菌生物膜在骨水泥表面形成。还研究并比较了载壳聚糖的 PMMA（相同的重量比）、载庆大霉素的 PMMA 和不含抗生素的 PMMA。选择了两种临床分离株，表皮葡萄球菌 389 和耐甲氧西林表皮葡萄球菌（MRSE287），以及两种标准菌株，表皮葡萄球菌（ATCC35984）和耐甲氧西林金黄色葡萄球菌（ATCC43300），使用平板扩散法、共聚焦激光扫描显微镜（CLSM）和扫描电子显微镜（SEM）在 6、12 和 24 小时评估细菌生物膜的形成。结果表明，26%HACC 载 PMMA 抑制了其表面的生物膜形成，而 PMMA 对照和载壳聚糖的 PMMA 则不能抑制生物膜形成。载庆大霉素的 PMMA 减少了耐甲氧西林金黄色葡萄球菌的活菌数，但抑制生物膜形成的能力低于 26%HACC 载 PMMA。实时 PCR 表明，26%HACC 载 PMMA 显著下调了编码多糖细胞间黏附（PIA）生物合成必需酶的 icaAD 的表达，上调了负调节 icaAD 表达的 icaR 的表达水平，还下调了编码已知与青霉素结合蛋白结合的膜结合酶的 mecA 的表达。我们的研究表明，26%HACC 载 PMMA 可防止包括抗生素耐药株在内的葡萄球菌在骨水泥表面形成生物膜，并下调抗生素耐药葡萄球菌的毒力相关基因表达，从而为防治植入物感染和骨髓炎提供了一种有前景的新策略。
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