牙周菌斑生物膜的体外模型建立

A pilot study of an model of periodontal biofilm in vitro

目的观察血链球菌、牙龈卟啉单胞菌和具核梭杆菌在人工牙根面上随培养时间变化形成单菌种、双菌种和三菌种生物膜的能力,期望在体外建立龈下菌斑生物膜模型。方法培养血链球菌、牙龈卟啉单胞菌和具核梭杆菌试验菌株,制备胶原包被羟磷灰石的人工牙根面,在人工牙根面上培养形成试验菌株的单菌种、双菌种和多菌种生物膜,并用扫描电镜观察三种试验菌株分别在培养24、48和72 h后形成单菌种、双菌种和三菌种生物膜的情况。结果单独培养24 h的血链球菌在人工牙根面上形成的完整生物膜,具备三维立体结构;培养48和72 h后,细菌密度逐渐增大,生物膜更加成熟。单独培养48 h的牙龈卟啉单胞菌形成的完整生物膜,具备三维立体结构;培养72 h后,细菌密度有所降低。牙龈卟啉单胞菌和具核梭杆菌培养24 h形成的完整的双菌种生物膜,具备三维立体结构;培养48和72 h后已看不出完整的生物膜结构,两菌的数量大幅度降低。血链球菌、牙龈卟啉单胞菌和具核梭杆菌在培养24 h时形成的较完整的三菌种生物膜,初步具备三维立体结构;三菌中,血链球菌、具核梭杆菌所占比列远大于牙龈卟啉单胞菌;培养48 h后,三菌种生物膜更加成熟,牙龈卟啉单胞菌所占比例有所增加,此时的生物膜已具备三维立体结构;培养72 h后,三菌种仍保持较完整的生物膜结构,但数量均有所下降。结论血链球菌、牙龈卟啉单胞菌和具核梭杆菌在培养24 h时间点已基本形成了完整的单菌种、双菌种和三菌种生物膜结构,故在今后建立龈下菌斑生物膜模型时可选取24 h这一时间点。

Objective The ability of the single-species and multi-species biofilm formation on the artificial root surface by Streptococcus sanguis （S.sanguiz）, Fasobacterium nucleatum （F.nucleatum） and Porphyromorms gingivalis fP.gingivalis） was observed in order to establish an in vitro subgingival plaque biofilm model. Methods Ssanguis, F..nucleatum and P.gingivalis were cultured, and collagen-treated hydroxyapatite was prepared as the artificial root surface. The formation of the single-species, dual-species and multi-species biofilms of S.sanguis, F.nucleatum and P.gingivalis on the artificial root surface were detected at 24, 48 and 72 h by scanning electron microscopy. Results S.sanguis biofilm was formed on the artificial root surface at 24 h, and showed a three-dimensional structure. The biofilm looked more mature at 48 and 72 h. P.gingivalis biofilm was formed at 48 h, and showed a three-dimensional structure. The density of P.gingivalis in the biofilm declined at 72 h. P.gingivalis and F.nucleaturn were able to form a complete dual-species biofilm with full three-dimensional structure at 24 h. The dualspecies biofilm of P.gingivalis and F.nucleatum lost the complete structure at 48 and 72 h, in which a significant reduction in the number of two types of bacteria was observed. S.sanguis, F..nucleatum and P.gingivalis formed a three-dimensional structure multi-species biofilm at 24 h. P.gingivalis shared lower proportion in the number of the three bacteria. The biofilm became more mature at 48 h. The proportion of P.gingivalis increased. At 72 h the biofilm remained a complete structure with a decline of the number of the three bacteria. Conclusion The single-species （S.sanguis or P.gingivalis）, dual-species（F.nucleatum and P.gingivalis） and multi-species（S.sanguis, F.nucleatum and P.gingivalis） biofilms were formed at 24, 48 and 72 h in this study. 24 hours may be an optimal period for establishing the biofilmmodel of periodontal bacteria for the further study.