# In Vitro Analysis of Organic Ester Functional Groups in Carious Dentine
## 1 Introduction  
Characterising and discriminating between denatured, necrotic, bacterially contaminated and demineralised tissue in a cavitated carious lesion is of clinical relevance when performing minimally invasive operative interventions [1, 2]. Carious dentine consists of two main histological zones [3, 4]. The outer, superficial, highly bacterially contaminated and denatured zone of tissue is clinically wet, soft and sticky--the caries-infected (bacterially contaminated) dentine. The remaining, deeper caries-affected (demineralised) dentine can be healed and repaired by the dentine-pulp complex and, therefore, can be retained and sealed off using biointeractive restorative materials [1, 5, 6, 7]. This selective, minimally invasive operative approach preserves more tissue and has been shown to improve the long-term survival of the dentine-pulp complex [1, 5, 7, 8]. The histological transition between these two zones of tissue, however, is diffuse and difficult to identifyclinically as well as in the laboratory, which leads to subjectivity and variations between operators/researchers and, therefore, often unnecessary excessive removal of tooth tissue by clinicians. Therefore, there is an unmet need to objectively delineate between caries-infected and affected dentine, marking the clinical endpoint of cautious dentine excavation in both laboratory investigations and the clinic [5; 9; 10]. In vitro studies have predominantly characterised various dentine tissues mechanically. [PERSON] and co-workers used microhardness to divide cautious dentine into outer (superficial) and inner lesion layers [11]. [PERSON] et al. (2010) further classified the carious dentine lesion according to its Knoop hardness value and its intrinsic autofluorescence characteristics, with caries-infected dentine (CID) <25 KHN and caries-affected dentine (CAD) between 25 and 40 KHN. However, the hardness measurement only indirectly represents the basic mechanical mineral integrity of the tissue, without a direct correlation with the biochemical characteristics of the lesion [12; 13], the latter having a role in tissue repair and biomaterial adhesion. Biochemically, carious dentine contains high levels of organic compounds, proportionate with a relatively lower inorganic content, compared to sound dentine [4; 14; 15]. [PERSON] et al. (2021) characterised the carious dentine lesion using the Raman spectroscopic protein/mineral ratio (amide I/phosphate) according to the tissue microhardness. That study concluded the amide I/phosphate Raman spectral ratio could be used as a biochemical method to characterise carious dentine with a direct association with lesion histology. In addition to amide I (-CO-NH-), which represents the main organic component in carious dentine, [PERSON] et al. (2014) showed that the protein ester functional group (-CO-OR-) can be considered a unique functional group in carious dentine, as it is not observed in sound dentine. This is due to the fact that esters are formed in an environment that contains less water and a low pH compared to that found in carious dentine [16]. However, its distribution has yet to be mapped through the histological zones of infected and affected dentine. Determining any difference in intensity, and therefore distribution, of the ester functional groups between infected and affected zones of carious dentine could help develop clinical indicators to aid practitioners with selective, minimally invasive operative carious dentine removal technologies and develop research towards developing novel chemical adhesion mechanisms for biointeractive restorative materials and tissue repair mechanisms. Therefore, this in vitro study aimed to compare the distribution of ester functional groups within carious dentine tissue (infected and affected dentine). The null hypothesis was that there were no differences in the Raman spectroscopic peak vibrational intensities of ester functional groups between infected and affected carious dentine tissues.