TITLE

DIAGNOSIS OF CARIES

DESCRIPTION

Technical field

The present invention relates to the diagnosis of caries in particular for the treatment of caries in dentine tissue

Background of the invention

In the treatment of carious lesions the major part of the carious dentine can be visually identified and be removed using mechanical force, i.e., dental drill tools. However, in all situations it might not be possible to remove all carious dentine tissue, but some carious dentine will remain and will become locked in underneath a filling used to repair the tooth. This will certainly cause a further development of carious dentine tissue, which can develop unseen due to the fact that it is hidden underneath the filling. And when wearer becomes aware of the carious dentine tissue it might be hard to save the tooth, but extraction or root filling may be needed.

This problem can of course be handled by excavating more dentine tissue from the tooth, but this on the other hand will lead to a weakening of the tooth strength (i.e., less crystals, hydroxyapatite, and less proteins, respectively), the crown walls will become too thin. Further this will lead to a decrease in tooth re-mineralisation since the healthy tissue is replaced by a non-living material like composite etc. Taking away living material would affect the whole tooth with both the inorganic and the organic parts, respectively.

Therefore, a chemical marker or method to identify carious dentine tissue may facilitate caries treatment and reduce the amount of healthy tooth tissue removed during treatment.

Dental caries is an endogenous infection of the calcified tissues of the teeth and is historical the consequence of the interaction between the oral microflora, the diet, the dentition and the oral environment [1]. The pathogenesis of dental caries is dependent upon the presence of fermentable sugars in the diet and the presence of cariogenic bacterial species [2-5]. The cariogenic micro organisms constitute a complex oral microflora with both acidogenic and aciduric properties [5]. When present in a metabolically active biofilm, covering the tooth surface, the underlying tooth surface may gradually become chemically modified which over time may result in a net loss of mineral.

From a clinical point of view, dental caries has been described as a soft, yellow-brownish discoloration of the dentine [6]. It has in vitro also been shown that dentine turns pale yellow when exposed to glucose [7]. Earlier investigations of dental caries have revealed reactions between proteins and sugars (generally called Maillard reactions) and it has been suggested that they are responsible for the typical discoloration [7]. Other authors have explained the discoloration as an effect of binding between keto groups of glyceraldehyde (a carbohydrate fermentation product) and carious dentine, for example, which would then cause the brown pigmentation [8]. This indicative discoloration also encompasses dentine when reacting with both glucose and glucose amine [8]. Furthermore, differences within carious tissues have been described by Kuboki and co-workers [9], who divided dental caries into a first/outer layer and a second/inner layer adjacent to normal dentine. The outer carious layer is more infected and necrotic, compared with the inner layer.

Carious dentine contains significant amounts of sugars [8] and can therefore form advanced glycation end products (AGEs), e.g. Maillard products, between sugars and the side chains of basic amino acids such as lysine and arginin [10]. These Maillard reactions are non-enzymatic crosslink reactions. The two most well-known Maillard reaction products found in carious dentine, pentosidine and carboxylmethyllysine [10, 11], have not been found in sound dentine [11].

In the prior art different methods for determining changes in teeth mineralization have been described. US-A1 -2005/0283058 thus discloses that early dental caries detection is carried out by a method that combines optical coherence tomography (OCT) and Raman spectroscopy to provide morphological information and biochemical specificity for detecting and characterizing incipient carious lesions found in extracted human teeth. OCT imaging of tooth samples demonstrated increased light back-scattering intensity at sites of carious lesions as compared to the sound enamel. Raman microspectroscopy and fibre-optic based Raman spectroscopy are used to characterize the caries further by detecting demineralization-induced alterations of enamel crystallite morphology and/or orientation.

Hynes, A. et al in BMC Medical Imaging 2005, 5:2, "Molecular mapping of periodontal tissues using infrared microspectroscopy" discloses use of infrared microspectroscopy to find pathogenic processes in periodontal tissue.

Antunes, A. et al in Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, August 2006, vol. 64, p. 1142-1146, "Spectroscopic alterations on enamel and dentin after nanosecond Nd:YAG laser irradiation" discloses that laser irradiation on hard tissue

produces a resistant surface that is likely to prevent caries. FTIR is thereby used to measure inorganic and organic compounds present in dental hard tissue by the KBr method.

Di Renzi, M. et al in Biomaterials, 2001 , vol. 22, p.787-792 discloses that photoacoustic FTIRS studies can be used chemical modifications in dentin surface.

Thus there is a need for a simple but safe method for diagnosing the presence of carious dentine tissue.

Summary of the present invention

The present invention relates to a method for diagnosing the optional presence of carious dentine tissue in a simple but reliable way. For that reason attempts of finding a specific group were performed.

Detailed description of the present invention

The present invention relates in particular to a specific chemical compound, an ester, which has been found present in carious dentine tissue. The chemical compound can be easily detected by infrared spectroscopy, or by fluorescent light, when a fluorescent moiety has been coupled to the chemical compound, in particular at the ester group.

In carious tissues, there might also be organic molecular alterations other than the Maillard reactions described above. The presence of esters in dental caries has been investigated and it has been found that esterases are more common in carious tissue than in intact tissue [12]. Furthermore, Dirksen [13] recognised esters, derived from bacterial lipid components such as cholesterol esters, in both sound and carious dentine.

However, experimental evidence of the presence of esters in dental caries is still lacking and possible differences in the esters for the outer and inner layers of dental caries have not been investigated. It can be hypothesised that the esterification of the hydroxyl groups of the acidic side chains of aspartate and glutamate residues facing an acidic environment (lactic acid) is unique to carious lesions. The aim of the present paper was therefore to evaluate the presence of the presumptive ester groups in the outer and inner layers of carious lesions.

Materials and Methods Sample preparation

The hard tissue from two human permanent premolars, with manifest carious lesions, was used for the experiments in the present study. The teeth were extracted due to the severe carious status and had no earlier dental restorations. The time between extraction and further handling was a maximum of one week. During this time, the teeth were stored in deionised water at +4°C.

The outermost part of the dental carious lesions was removed with a sterilised excavator and thrown away. The remaining dental caries was divided into two layers; one outer layer with discoloured, soft and infected dental caries and one inner layer with harder, less discoloured dental caries. The layers of outer and inner dental caries were removed with an excavator and put into test tubes. A clean excavator was used for the different respective samples, i.e., a clean excavator was used for each layer of the tooth. Sound dentine was excavated from an uninfected part of the tooth, as a reference for unaltered peptides.

This excavating procedure was repeated for the second tooth. After excavation, the selected tooth material was left spontaneously to dry at ambient temperature. The dry weight of each tooth sample was approximately 1 mg.

Fourier Transform Infrared Spectroscopy (FTIR) The tooth material that had been removed was mixed with potassium bromide (KBr) for KBr-pellet preparation and subsequent FTIR examination. The total weight of each KBr pellet was 100 mg. The IR analyses were performed using a Mattson Cygnus 100 FTIR spectrophotometer with 4 cm ^"1 resolution. Each spectrum was acquired from about 100 scans. To remove the effects of atmospheric carbon dioxide and water vapour, the instrument was purged with analytical instrument quality air, dried and purified with a

Balstron type 75-60 conditioner. All FTIR spectra were acquired within a few hours of KBr- pellet preparation: most immediately after production of the KBr pellet. The spectra were baseline corrected using the FTIR software. For all spectra, the same wave-number positions were chosen.

A summary of the FTIR spectra with the different assignments of sound dentine and carious tissues is presented in Figure 1 and Table 1 respectively. None of the samples exhibited a distinct absorption peak at 1735-1750 cm ^"1 , the characteristic peak of the carbonyl group of esters [14]. However, one knee around 1740 cm ^"1 of the amide I band at 1660 cm ^"1 could be detected for both the inner layer dental caries samples and one of the outer layer dental caries samples (Figure 2). This knee was not present in the dentine spectra (Figure 3). Neither sound dentine nor carious tissue exhibited a distinct peak at

1050-1300 cm ^'1 , another characteristic IR absorption region of esters [15]. However, the peak at 1030 cm ^"1 for sound dentine was shifted to approximately 1040 cm ^'1 for the carious samples (Figure 4).

Table 1. Summary of suggested assignments [14-16] for the major FTIR peaks detected for healthy and carious dentine.

Graph Tooth Tooth Positions (cm ^'1 ) of major peaks with suggested assignments notasample no. N-H C-H C=O C=O P-OH P-O tion stretch stretch of stretch of bend deformaesters amide I tion

A Healthy 1 3500- 2927 - 1668 1032 561 dentine 3380

B Healthy 2 3500- 2941 _ 1653 1034 561 dentine 3380

C Outer 1 3500- 2920 Knee at 1655 1045 561 carious 3380 1740 cm ^'1 layer

D Outer 2 3500- 2924 - 1653 1034 561 carious 3380 layer

E Inner 1 3500- 2920 Knee at 1657 1038 563 carious 3380 1740 cm ^"1 layer

F Inner 2 3500- 2924 Knee at 1653 1034 559 carious 3380 1740 cm ^"1 layer

The results clearly show that hexose ester groups are unique to carious tissue and are therefore not found in sound dentine.

The majority of known analytical techniques require that proteins are extracted from the calcified tissue in order to enable analysis of functional groups, e.g. esters. The first step is mechanically to turn the tooth tissue into pieces; the dentine needs to be pulverised and dental caries only needs to be excavated with sharp excavators for the subsequent extraction of the proteins [18]. Furthermore, after the mechanical treatment, the solubilisation of human dental collagen requires a combination of acids and enzymes [18, 19]. To circumvent the difficulties involved in extracting proteins from dentine and dentine caries, FTIR was selected in the present paper, since it is a technique that permits the analysis of solid material without extensive sample preparation.

FTIR has frequently been used in dental research and, when studying the FTIR spectra of healthy dentine presented in earlier studies [17, 20-25], no knee around 1740 cm ^"1 at the amide I band can be detected, which on the other side have been found in the present study. The second characteristic absorption region of esters at 1050-1300 cm ^'1 is more

SUBSTITUTE SHEET (RULE 28))

difficult to interpret, since other functional groups also absorb in that region. Even though no distinct peaks could be found for sound dentine or carious samples in that region, the slight shift in the peak at 1040 cm ^"1 could possibly have been caused by esters. However, although FTIR has frequently been used for studies of dentine, there is a lack of published FTIR spectra of carious dentine.

The results herein clearly indicate the presence of ester groups in both inner layer dental caries samples and in only one of the outer dental caries samples. Thus the results show that hexose ester groups are unique to carious tissue and are therefore not found in healthy dentin. This result can possibly be explained by the fact that an acidic environment is required for ester groups to be formed. Carboxylic acids react with small carbohydrates to form esters through a condensation reaction known as esterification [14]. Furthermore, by removing water, more esters are formed [14]. Mineralised tissue contains less water than saliva, which is a condition for esters. When formed, the stability of esters varies with pKa values from 11 to 25, depending on the groups/atoms flanking the ester [26]. In addition, when esters are produced in vitro in transesterification reactions, they are stable at pH 4-8. Furthermore, they are not degraded, since the degrading enzyme lipase does not exist in the oral cavity [27]. In a carious lesion, the outer part is older and has a higher pH [6], since it is more exposed to the neutral pH of saliva, compared with the inner layer of dental caries. A higher pH in the outer layer of dental caries could therefore possibly cause a decrease in the stability of esters, as indicated by the results in the present study (Figure 2).

Another significant feature of dental carious lesions is the arrest which has been suggested to be caused by Maillard reactions [8, 11]. These findings have been confirmed by fluorescence measurements of the Maillard products from collagen digests of carious tissue [11]. Furthermore, the Maillard products modify the basic amino groups in collagen, which convert the dentine to a collagenase-resistant form [8]. In addition, Armstrong [8] has claimed that the hydroxyl groups are acetylated in dentine collagen, which modifies collagen to a collagenase-resistant form [8].

In a further test dentine samples were placed in lactic acid (0.4 M) without normal dentinal bacteria and with added D-glucose (0.2 M) (Fig. 5) or dentine sample in lactic acid (0.4 M) with normal dentinal bacteria and with added D-glucose (0.2 M) (Fig.6) and these were analysed with FTIR and compared to a dentine spectrum with no added D-glucose (reference, used as the subtraction spectra).

Since the amount of carbonyl groups seemed small Fouir Self-Deconvolution technique was used in order to enhance the presence of carbonyl bands in the spectra. (Fig. 5 and 6)

As a result of the deconvolution peaks appear at 1725 cm ^"1 and 1720 cm ^"1 , respectively. In avoiding false peaks that easily may appear when deconvolution is performed spectrum subtraction techniques was also used (Fig. 7 and Fig. 8). A spectrum of dentine in lactic acid with a proposed normal bacteria content and with no additional D-glucose was used as reference i.e. a carbonyl free spectrum.

Peak appearance is due to the addition of sugar for sample in Fig. 5. In addition the normal dentine bacteria seemed to have less effect on the carbonyl ester formation. Note, no caries bacteria are used in this study.

Peak appearance is due to the addition of sugar for sample in Fig. 6. Again, the normal dentine bacteria seemed to have less effect on the carbonyl ester formation. Note, no caries bacteria are used in this study.

FIGURE LEGENDS

Figure 1. FTIR spectra of A) healthy dentine of tooth 1 , B) healthy dentine of tooth 2, C) outer layer caries of tooth 1 , D) outer layer caries of tooth 2, E) inner layer caries of tooth 1 and F) inner layer caries of tooth 2.

Figure 2. The amide I band of carious tissue from outer (graphs C and D) and inner (graphs E and F) layers of the lesions. The knees at 1740 cm ^'1 are marked with arrows.

Figure 3. The amide I band of sound dentine from three different teeth; A) sound dentine of tooth 1 , B) sound dentine of tooth 2 and *) origins from earlier experiments [17].

Figure 4. A zoom of the region between 1400 and 1000 cm ^"1 , demonstrating a slight shift at 1040 cm-1 for carious samples. FTIR spectra of A) sound dentine of tooth 1 , B) healthy dentine of tooth 2, C) outer layer caries of tooth 1 , D) outer layer caries of tooth 2, E) inner layer caries of tooth 1 and F) inner layer caries of tooth 2.

Figure 5: A deconvoluted spectra of dentine without normal dentine bacteria in lactic acid and also with added glucose. (1725 cm ^"1 ).

Figur 6: A deconvoluted spectra of dentine which includes the normal dentine bacteria in lactic acid and also with added glucose. (1720 cm ^"1 ).

Figur 7: The sample in Fig. 5 (A deconvoluted spectra of dentine without normal dentine bacteria in lactic acid + glucose) minus a deconvoluted spectra of dentine including normal dentine bacteria in lactic acid (set as the reference).

Figure 8: The sample in Fig. 6 (A deconvoluted spectra of dentine which includes the normal dentine bacteria in lactic acid in lactic acid + glucose) minus a deconvoluted spectra of dentine including normal dentine bacteria in lactic acid (set as the reference).

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