Department of Restorative Dentistry, King's College London, London, UK; Department of Microbiology, Dental Institute, King's College London, London, UK.
Int Endod J. 2014 Aug;47(8):756-68. doi: 10.1111/iej.12214. Epub 2014 Jan 9.
To establish a nutrient-stressed multispecies model biofilm and investigate the dynamics of biofilm killing and disruption by 1% trypsin and 1% proteinase K with or without ultrasonic activation.
Nutrient-stressed biofilms (Propionibacterium acnes, Staphylococcus epidermidis, Actinomyces radicidentis, Streptococcus mitis and Enterococcus faecalis OMGS 3202) were grown on hydroxyapatite discs and in prepared root canals of single-rooted teeth in modified fluid universal medium. The treatment groups included trypsin, proteinase K, 0.2% chlorhexidine gluconate and 1% sodium hypochlorite (NaOCl) (with and without ultrasonics). NaOCl and chlorhexidine were the positive controls and untreated group, and sterile saline was the negative control. The biofilms were investigated using confocal laser scanning microscopy (CLSM) with live/dead staining and quantitative microbial culture.
Nutrient stress in the multispecies biofilm was apparent as the medium pH became alkaline, glucose was absent, and serum proteins were degraded in the supernatant. The CLSM showed the percentage reduction in viable bacteria at the biofilm surface level due to nutrient starvation. On the disc model, trypsin and proteinase K were effective in killing bacteria; their aerobic viable counts were significantly lower (P < 0.01) than the negative control and chlorhexidine. NaOCl was the most effective agent (P < 0.001). In the tooth model, when compared to saline, trypsin with ultrasonics caused significant killing both aerobically and anaerobically (P < 0.05). Chlorhexidine (1.46 ± 0.42), trypsin (3.56 ± 1.18) and proteinase K (4.2 ± 1.01) with ultrasonics were significantly effective (P < 0.05) in reducing the substratum coverage as compared to saline with ultrasonics (12% ± 4.9).
Trypsin with ultrasonic activation has a biofilm killing and disrupting potential.
建立营养胁迫的多菌种模型生物膜，并研究 1%胰蛋白酶和 1%蛋白酶 K 在有无超声激活的情况下对生物膜杀伤和破坏的动力学。
将营养胁迫的生物膜（痤疮丙酸杆菌、表皮葡萄球菌、放射形放线菌、口腔链球菌和 OMGS 3202 粪肠球菌）种植在羟基磷灰石盘上，并在经过改良的通用培养液中于单根牙的预备根管内生长。实验组包括胰蛋白酶、蛋白酶 K、0.2%葡萄糖酸氯己定和 1%次氯酸钠（有和没有超声）。次氯酸钠和氯己定作为阳性对照，未处理组作为阴性对照，无菌生理盐水作为阴性对照。使用带有活/死染色和定量微生物培养的共聚焦激光扫描显微镜（CLSM）对生物膜进行研究。
多菌种生物膜中的营养胁迫表现为培养基 pH 值变为碱性、葡萄糖不存在以及上清液中血清蛋白被降解。CLSM 显示由于营养饥饿，生物膜表面水平的活菌百分比减少。在圆盘模型中，胰蛋白酶和蛋白酶 K 能够有效地杀死细菌；与阴性对照和氯己定相比，它们的需氧活菌计数明显较低（P<0.01）。次氯酸钠是最有效的试剂（P<0.001）。在牙齿模型中，与生理盐水相比，超声作用下的胰蛋白酶会导致需氧和厌氧条件下的显著杀伤（P<0.05）。与生理盐水相比，超声作用下的氯己定（1.46±0.42）、胰蛋白酶（3.56±1.18）和蛋白酶 K（4.2±1.01）在降低基质覆盖率方面具有显著的效果（P<0.05）。
超声激活的胰蛋白酶具有生物膜杀伤和破坏的潜力。
