Evaluating models and assessment techniques for understanding oral biofilm complexity

Oral biofilms are three-dimensional (3D) complex entities initiating dental diseases and have been evaluated extensively in the scientific literature using several biofilm models and assessment techniques. The list of biofilm models and assessment techniques may overwhelm a novice biofilm researcher. This narrative review aims to summarize the existing literature on biofilm models and assessment techniques, providing additional information on selecting an appropriate model and corresponding assessment techniques, which may be useful as a guide to the beginner biofilm investigator and as a refresher to experienced researchers. The review addresses previously established 2D models, outlining their advantages and limitations based on the growth environment, availability of nutrients, and the number of bacterial species, while also exploring novel 3D biofilm models. The growth of biofilms on clinically relevant 3D models, particularly melt electrowritten fibrous scaffolds, is discussed with a specific focus that has not been previously reported. Relevant studies on validated oral microcosm models that have recently gaining prominence are summarized. The review analyses the advantages and limitations of biofilm assessment methods, including colony forming unit culture, crystal violet, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt assays, confocal microscopy, fluorescence in situ hybridization, scanning electron microscopy, quantitative polymerase chain reaction, and next-generation sequencing. The use of more complex models with advanced assessment methodologies, subject to the availability of equipment/facilities, may help in developing clinically relevant biofilms and answering appropriate research questions.