## 4 Discussion  
A recent systematic review by [PERSON] et al. shed light on the lack of standardization in _in-vitro_-studies investigating the cleaning and disinfection of root canals [34]. An uneven contact time of irrigants with the root canal surface, differences in flow rate of the disinfect and a variable volume used for irrigation represent imopardeables and can alter the reduction of debris [40]. Another point of criticism was a lack of documentation of the power settings for the devices used for activation. Furthermore, it is worth considering the dimensions of the fluid reservoir above the root canal representing the access cavity as well as the detection limit for debris quantification, which may affect the outcomes. Therefore, a major goal of this study was to develop a model system that provides a high level of standardization. The intention was to keep the experimental focus on the research question, whereas uncertainties due to anatomy or debris accumulation, as well as variabilities of the operator in irrigation and activation, should be minimized. A newly developed, 3D-printed model was used to fasten the roots during processing, which offered the benefit of a standardized reservoir in size of a molar's access cavity that held a defined liquid volume. Differences in irrigation by anatomical variations of the endodontic access in natural teeth were thus avoided. A syringe pump provided a precise flow of liquids during root canal instrumentation and activation sequences. Hose connectors in the 3D-printed device offered the possibility to rinse the samples during preparation and activation without use of a needle. Thus, alterations by irrigation needles were averted in the SAI and LAI groups. Another uncertainty is the formation and accumulation of hard tissue debris in root canals. Preliminary experiments have shown that hard tissue debris accumulates unpredictably and forms to a relevant degree when irrigation is kept artificially low [1; 32]. However, a thorough disinfection of the root canals during the course of root canal instrumentation prevents accumulation of debris, at least _in vitro_, as shown in this study. This corresponds to _in-vivo_-data, describing the absence of hard tissue debris after root canal preparation with ample irrigation [41]. Another reason for the lack of AHTD could be the root canal preparation technique, as rotating multi-file systems, which implement  
Figure 5: (**a**) Median volumes with 25–75% quantiles of artificial dentine debris in the coronal, middle and apical third of the root samples. (**b**) Median debris volume with 25–75% quantiles after final irrigation at all levels of the root canal. Asterisks denote statistical differences between groups. a crown-down technique, are known to produce less debris compared to single-file systems [42]. Although these findings highlight the importance of intensive irrigation during instrumentation, an evaluation of activation methods based on those minute amounts of debris is not reasonable. One corrective measure suggested in the literature is the reduction of irrigation efforts to a minimum and, thus, provoking debris formation [1, 32]. However, this procedure differs from clinical recommendations, and a standardization of debris formation is not possible. To overcome variations in debris formation and provide similar baseline conditions before final irrigation, an artificial hard tissue debris based on human dentine mixed with sodium hypochlorite and BaSO4 was fabricated, which has not been described before. The focus was to reproduce the consistency of natural AHTD and to achieve similar chemo-mechanical characteristics in the root canal. Previously, research groups used artificial debris compounded with sodium hypochlorite in a \"root-canal-grove model\" [43, 44, 45]. However, this preclinical model was based on longitudinally dissected teeth with simple root canal morphology that does simulate complex root canal configurations like in mandibular molars.