EP2079357A1 - Diagnosis of caries - Google Patents
Diagnosis of cariesInfo
- Publication number
- EP2079357A1 EP2079357A1 EP07835124A EP07835124A EP2079357A1 EP 2079357 A1 EP2079357 A1 EP 2079357A1 EP 07835124 A EP07835124 A EP 07835124A EP 07835124 A EP07835124 A EP 07835124A EP 2079357 A1 EP2079357 A1 EP 2079357A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- dentine
- carious
- caries
- tooth
- tissue
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 208000002925 dental caries Diseases 0.000 title description 49
- 238000003745 diagnosis Methods 0.000 title description 3
- 210000004268 dentin Anatomy 0.000 claims abstract description 74
- 238000000034 method Methods 0.000 claims abstract description 17
- 238000010521 absorption reaction Methods 0.000 claims abstract description 6
- 238000004566 IR spectroscopy Methods 0.000 claims abstract description 3
- -1 hexose ester Chemical class 0.000 claims description 4
- 229930182478 glucoside Natural products 0.000 claims 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 22
- 150000002148 esters Chemical class 0.000 description 17
- 238000001228 spectrum Methods 0.000 description 16
- 241000894006 Bacteria Species 0.000 description 13
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 11
- 239000004310 lactic acid Substances 0.000 description 11
- 235000014655 lactic acid Nutrition 0.000 description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 10
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 10
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 10
- 102000008186 Collagen Human genes 0.000 description 8
- 108010035532 Collagen Proteins 0.000 description 8
- 229920001436 collagen Polymers 0.000 description 8
- 210000003127 knee Anatomy 0.000 description 7
- 108010005094 Advanced Glycation End Products Proteins 0.000 description 6
- 238000001157 Fourier transform infrared spectrum Methods 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 235000000346 sugar Nutrition 0.000 description 6
- 150000001408 amides Chemical class 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 150000008163 sugars Chemical class 0.000 description 5
- 230000002378 acidificating effect Effects 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 4
- 238000002845 discoloration Methods 0.000 description 4
- 125000004185 ester group Chemical group 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 230000004075 alteration Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 210000003298 dental enamel Anatomy 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000011049 filling Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000012014 optical coherence tomography Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 238000004971 IR microspectroscopy Methods 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 238000001069 Raman spectroscopy Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 230000001013 cariogenic effect Effects 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 238000005115 demineralization Methods 0.000 description 2
- 230000002328 demineralizing effect Effects 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 230000037213 diet Effects 0.000 description 2
- 230000032050 esterification Effects 0.000 description 2
- 238000005886 esterification reaction Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 244000005706 microflora Species 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 230000003239 periodontal effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 210000003296 saliva Anatomy 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- LCTORNIWLGOBPB-GASJEMHNSA-N (3r,4s,5s,6r)-2-amino-6-(hydroxymethyl)oxane-2,3,4,5-tetrol Chemical compound NC1(O)O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O LCTORNIWLGOBPB-GASJEMHNSA-N 0.000 description 1
- HZDLKNOGBSMKRN-UHFFFAOYSA-N 2-amino-5-[[5-oxo-4-(2,3,4-trihydroxybutyl)-1,4-dihydroimidazol-2-yl]amino]pentanoic acid Chemical compound OC(=O)C(N)CCCNC1=NC(CC(O)C(O)CO)C(=O)N1 HZDLKNOGBSMKRN-UHFFFAOYSA-N 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N Arginine Chemical compound OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- MNQZXJOMYWMBOU-VKHMYHEASA-N D-glyceraldehyde Chemical compound OC[C@@H](O)C=O MNQZXJOMYWMBOU-VKHMYHEASA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 238000001530 Raman microscopy Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002053 acidogenic effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229940009098 aspartate Drugs 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 210000004763 bicuspid Anatomy 0.000 description 1
- 230000033558 biomineral tissue development Effects 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 150000001840 cholesterol esters Chemical class 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 210000004513 dentition Anatomy 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000002059 diagnostic imaging Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-L glutamate group Chemical group N[C@@H](CCC(=O)[O-])C(=O)[O-] WHUUTDBJXJRKMK-VKHMYHEASA-L 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 125000000468 ketone group Chemical group 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- VUZPPFZMUPKLLV-UHFFFAOYSA-N methane;hydrate Chemical compound C.O VUZPPFZMUPKLLV-UHFFFAOYSA-N 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- AYEKKSTZQYEZPU-RYUDHWBXSA-N pentosidine Chemical compound OC(=O)[C@@H](N)CCCCN1C=CC=C2N=C(NCCC[C@H](N)C(O)=O)N=C12 AYEKKSTZQYEZPU-RYUDHWBXSA-N 0.000 description 1
- 230000019612 pigmentation Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000036346 tooth eruption Effects 0.000 description 1
- 238000005809 transesterification reaction Methods 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B6/00—Apparatus or devices for radiation diagnosis; Apparatus or devices for radiation diagnosis combined with radiation therapy equipment
- A61B6/50—Apparatus or devices for radiation diagnosis; Apparatus or devices for radiation diagnosis combined with radiation therapy equipment specially adapted for specific body parts; specially adapted for specific clinical applications
- A61B6/51—Apparatus or devices for radiation diagnosis; Apparatus or devices for radiation diagnosis combined with radiation therapy equipment specially adapted for specific body parts; specially adapted for specific clinical applications for dentistry
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
- A61B5/0082—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence adapted for particular medical purposes
- A61B5/0088—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence adapted for particular medical purposes for oral or dental tissue
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61C—DENTISTRY; APPARATUS OR METHODS FOR ORAL OR DENTAL HYGIENE
- A61C3/00—Dental tools or instruments
Definitions
- the present invention relates to the diagnosis of caries in particular for the treatment of caries in dentine tissue
- 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.
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Dentistry (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medical Informatics (AREA)
- Engineering & Computer Science (AREA)
- Pathology (AREA)
- Surgery (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Heart & Thoracic Surgery (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- High Energy & Nuclear Physics (AREA)
- Audiology, Speech & Language Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Optics & Photonics (AREA)
- Radiology & Medical Imaging (AREA)
- Cosmetics (AREA)
- Dental Tools And Instruments Or Auxiliary Dental Instruments (AREA)
- Measurement Of The Respiration, Hearing Ability, Form, And Blood Characteristics Of Living Organisms (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
Abstract
The present invention relates to a method for determining the presence of carious tissue in dentine by identifying by means of infrared spectroscopy any absorption at the wavelength 1740 cm-1.
Description
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 "1and 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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Claims
1. Method for determining the presence of carious tissue in dentine by identifying by means of infrared spectroscopy any absorption at the wavelength 1740 cm'1.
2. Method according to claim 1 wherein any absorption at the wavelength 1040 cm"1 is determined, as well
3. Method according to claim 1 , wherein a hexose ester is determined.
4. Method according to claim 3, wherein a glucoside ester is determined.
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