# In Vitro Analysis of Organic Ester Functional Groups in Carious Dentine
## 4 Discussion  
Contemporary various dentine excavation should be selective, preserving the remineralisable anxious tissues and removing the infected and necrotic tissue only [1, 6]. Thus, clinicians and researchers are continually trying to develop and optimise technologies that can objectively discriminate between these histological carions dentine zones. Biochemical alterations have been shown to play a significant role in carions dentine, specifically the organic components, with regard to tissue repair [14, 21, 22]. In vitro studies have used the organic components to distinguish carions dentine, as they become more exposed after demineralisation [14, 20, 23, 24]. Specifically, the ester functional group has been isolated in carions dentine but not in sound dentine [18, 25]. However, its distribution has not been characterised histologically to differentiate between carions dentine zones. Esters are naturally formed from reactions between structures with alcohol functions, such as carbohydrates and carboxylic acids, from protonated proteins and are catalysed by acidic agents, described as Fischer esterification. This process is influenced by water levels and changes in acidity, which means that it increases in an environment that contains low water content and low pH [16]. The present study demonstrated that the increased relative intensity of ester functional groups was higher in caries-infected dentine compared to the adjacent caries-affected dentine. This finding is consistent with the established knowledge that there are more proteins in infected dentine compared to sound tissue [14, 21]. In addition, it is in agreement with the nature of ester formation, as it forms in a more acidic environment, such as that found in caries-infected dentine. However, in the present study, there was a trace amount of ester function found in sound dentine (Table 1). This could be explained by the fact that the spectral data of the vibration bands of ester functional groups do not differentiate between types of esters, which also include glycerol-based phospholipids such as cholesterol- and phosphatidyl esters, a normal component of cell membranes [26, 27]. Not all types of esters in carions dentine zones have been characterised. Only carbonyl ester functions (COOR) have been identified in carions tissues ([PERSON] et al., 2014, 2017) [14, 18]. Besides these carbonyl ester functions (COOR), there might also be phosphor esters from lipids [28, 29], trans- or diesters and from sugar acetals [30] or cyclic esters that can arise from lactones (lactic acid esters) originating from carionic bacteria [31, 32]. In the present study, Raman microscopy detected all vibrational bands of ester functional groups. Therefore, a detailed analysis of ester functional groups in each carions dentine zone could be investigated further in the future. Figure 2: (A) A photomicrograph of one of the samples after sectioning and (B) the direction line of the Raman point scans that were taken to calculate the intensity of ester functional groups. LY was used in this study because it can react covalently with all ester groups [18]. In the present study, a change in the colour of the curious dentine was subjectively observed, which is explained by the fact that significant amounts of ester functional groups in infected and affected dentine visibly reacted with LY, but no changes in sound tissues were perceived. To the authors' knowledge, the present study is the first to objectively show the concentration and distribution of ester functional groups within carious dentine tissues, highlighting this important biochemical alteration and correlating it to the histological zones of caries-infected and caries-affected dentine. This, in turn, could pave the way for the investigation of objective clinical markers to delineate these zones of carious dentine to aid the clinician in their selective removal as part of the minimally invasive operative approach to deep cavitated lesions.