# A Novel Dental Caries Model Replacing, Refining, and Reducing Animal Sacrifice
## 4 Discussion  
An innovative in vitro caries model was developed herein aiming to decrease unnecessary animal sacrifice. Caries-promoting conditions mimicking in vivo caries models in murine were used to induce reproducible carious lesions within 5 days in extracted jaws of healthy animals sacrificed for other experiments and were no longer needed. To ensure the reliability of the in vitro development process, a resembling in vivo model study was conducted, and carious lesions were compared using qualitative and quantitative methods in both models. In the in vitro model, reproducible caries lesions were induced six times faster than in the in vivo model without compromising the data that can be drawn from the experimental conditions and without additional animal death. Caries lesions treatment requires the removal of the infected hard dental tissues, and restoring the lost tissues using different types of restorative dental materials, mainly by amalgam, composite resin or glass-ionomer cement. Caries removal was mostly done by traditional low-speed and high-speed rotatory instruments. Recently, several innovative techniques have been suggested for minimal-invasive caries removal: sonic and ultrasonic oscillating devices [22], lasers [23], and chemo-mechanical solutions [24]. Caries modeling is necessary for evaluating innovative restorative and removal materials before clinical trials. [PERSON] et al. [25] have reviewed in vitro biofilm models to study dental caries. They described the high variability and complexity of in vitro models; numerous models were conducted over human teeth or enamel slabs. Demineralization is very difficult to diagnose and quantify using these models, and it often requires unique equipment with a highly sensitive technique, such as polarized light microscopy [26; 27], microhardness [28; 29], atomic force microscopy [30], or quantitative light fluorescence (QLF) [31; 32]. Numerous caries studies have tried to quantify carious lesions development in vivo. [PERSON] [14] was the first to describe caries-scoring over hemi-sections rat molars. His scoring method, using murexide dye, is a common method still in use [33; 34; 35]. [PERSON] et al. [34] described the radiographic analysis of caries induced in rats; \(\mu\)CT scans were used to compare the mineral density of rats' molar enamel after calibration with hydroxyapatite standards of appropriate density. To our knowledge, [PERSON] et al. [15] described exclusively the use of  
Figure 9: Cariogenic diet and bacterial infection increase the relative percentage of _S. mutans_ from total bacterial counts in the mice oral habitat. Cariogenic (high-sucrose) diet and bacterial infections induced a significant increase of _S. mutans_ bacterial load. The results are presented as percentages normalized to the total bacterial count in each group. Blue = total bacteria viable count. Orange =_S. mutans_ viable count. [PERSON]'s modification [36] for [PERSON] caries scoring method in a murine mice model; all mandibular and maxillary molars were scored for smooth surface and sulcal caries. The clinical and radiographic demonstrations of the carious lesions evolved in the models described in the present study enables the identification of the lesion, quantification of the demineralization, and characterization in a similarity to the identification of carious lesions by dentists. Furthermore, the methods described require only stereomicroscope and \(\upmu\)CT, the analysis is relatively simple, and the results are repeatable. The demineralization process is dependent on the surface width and morphology. Murine molar teeth morphology contain sulci and fissures [37], which mimics the human molar morphology, but they are smaller, and the dental hard tissues are thinner than humans. Hence, the caries lesion development in the murine teeth is accelerated compared to the process in human teeth or enamel slabs [38]. For example, [PERSON] et al. [31] showed initial caries lesions over human molar enamel blocks after 30 days in caries-promoting conditions.