Chen Xi, Hirt Helmut, Li Yuping, Gorr Sven-Ulrik, Aparicio Conrado
Minnesota Dental Research Center for Biomaterials and Biomechanics, Department of Restorative Sciences, University of Minnesota School of Dentistry, Minneapolis, Minnesota, United States of America.
Department of Diagnostic and Biological Sciences, University of Minnesota School of Dentistry, Minneapolis, Minnesota, United States of America.
PLoS One. 2014 Nov 5;9(11):e111579. doi: 10.1371/journal.pone.0111579. eCollection 2014.
Infection is one of the most prevalent causes for dental implant failure. We have developed a novel antimicrobial peptide coating on titanium by immobilizing the antimicrobial peptide GL13K. GL13K was developed from the human salivary protein BPIFA2. The peptide exhibited MIC of 8 µg/ml against planktonic Pseudonomas aeruginosa and their biofilms were reduced by three orders of magnitude with 100 µg/ml GL13K. This peptide concentration also killed 100% of Streptococcus gordonii. At 1 mg/ml, GL13K caused less than 10% lysis of human red blood cells, suggesting low toxicity to mammalian cells. Our GL13K coating has also previously showed bactericidal effect and inhibition of biofilm growth against peri-implantitis related pathogens, such as Porphyromonas gingivalis. The GL13K coating was cytocompatible with human fibroblasts and osteoblasts. However, the bioactivity of antimicrobial coatings has been commonly tested under (quasi)static culture conditions that are far from simulating conditions for biofilm formation and growth in the oral cavity. Oral salivary flow over a coating is persistent, applies continuous shear forces, and supplies sustained nutrition to bacteria. This accelerates bacteria metabolism and biofilm growth. In this work, the antimicrobial effect of the coating was tested against Streptococcus gordonii, a primary colonizer that provides attachment for the biofilm accretion by P. gingivalis, using a drip-flow biofilm bioreactor with media flow rates simulating salivary flow. The GL13K peptide coatings killed bacteria and prevented formation and growth of S. gordonii biofilms in the drip-flow bioreactor and under regular mild-agitation conditions. Surprisingly the interaction of the bacteria with the GL13K peptide coatings ruptured the cell wall at their septum or polar areas leaving empty shell-like structures or exposed protoplasts. The cell wall rupture was not detected under regular culture conditions, suggesting that cell wall rupture induced by GL13K peptides also requires media flow and possible attendant biological sequelae of the conditions in the bioreactor.
感染是牙种植体失败最常见的原因之一。我们通过固定抗菌肽GL13K在钛表面开发了一种新型抗菌肽涂层。GL13K是从人唾液蛋白BPIFA2衍生而来。该肽对浮游铜绿假单胞菌的最低抑菌浓度为8μg/ml,100μg/ml的GL13K可使其生物膜减少三个数量级。这个肽浓度也能杀死100%的戈登链球菌。在1mg/ml时,GL13K对人红细胞的裂解率低于10%,表明对哺乳动物细胞毒性较低。我们的GL13K涂层此前也已显示出对种植体周围炎相关病原体(如牙龈卟啉单胞菌)的杀菌作用和对生物膜生长的抑制作用。GL13K涂层与人成纤维细胞和成骨细胞具有细胞相容性。然而,抗菌涂层的生物活性通常是在(准)静态培养条件下测试的,这些条件远不能模拟口腔中生物膜形成和生长的条件。口腔唾液在涂层上的流动是持续的,施加连续的剪切力,并为细菌提供持续的营养。这加速了细菌代谢和生物膜生长。在这项工作中,使用模拟唾液流动的介质流速的滴流生物膜生物反应器,测试了该涂层对戈登链球菌的抗菌效果,戈登链球菌是一种主要的定植菌,为牙龈卟啉单胞菌生物膜的积聚提供附着。GL13K肽涂层在滴流生物反应器中和常规轻度搅拌条件下杀死细菌,并阻止戈登链球菌生物膜的形成和生长。令人惊讶的是,细菌与GL13K肽涂层的相互作用使细胞壁在其隔膜或极性区域破裂,留下空壳状结构或暴露的原生质体。在常规培养条件下未检测到细胞壁破裂,这表明GL13K肽诱导的细胞壁破裂也需要介质流动以及生物反应器中条件可能伴随的生物学后果。