Corresponding author: Maryan Lelyukh (
At the present stage of development of medical science, experimental and clinical dentistry is often used in scientific research, which allows diagnosing preclinical stages of manifestations of changes in tissues and organs of the oral cavity and biological fluids at the level of nanotechnology enabling to study the mechanisms of molecular transformation that lead to occurrence of pathological changes. One of the promising methods of research is infrared spectroscopy. The advantage of the method is that the absorption spectrum can be obtained with a small amount of the test substance (0.1–0.01 cm3) and under conditions of different aggregate states (solution, powder, liquid).
Infrared spectroscopy (
The molecular structure of apatites – mineralized tissues of the tooth, can differ sharply both in the conditions of the physiological norm and in pathological conditions. The optimal composition of the hydroxyapatite corresponds to the formula Ca10 (PO4)6 (OH)2 with a molecular ratio of Ca/P ions corresponding to 1.67 (
It is established that the nature of chemical bonds in the enamel structure allows determining the initial changes that lead to a violation of the stoichiometry of hydroxyapatite crystals, as well as its demineralization and recrystallization (
There are two types of oscillations of the molecules: valence (oscillations of atoms occur along the axis of the valence bond) and deformation (the atoms in the oscillations deviate from the axis of the valence bond). For each chemical group, there are the corresponding oscillation frequencies (wave numbers) and absorption bands (characteristics). On the spectra, fluctuations of structural molecular and ionic units of the inorganic matrix such as phosphate, carbonate, and hydrophosphate ions are recorded, as well as numerous oscillations associated with a protein matrix. The spectrum band is described by the following indicators: intensity, width, type of polarization (
The main aim of the work was to investigate the enamel discoloration of vital teeth and to determine the effect of remineralizing therapy on valence grouping of the enamel surface of the experimental teeth with the help of the active ingredient of carbamide peroxide (
As a result of analysis of scientific sources, short reports, patents and own research (
carbamide peroxide – 30
glycerol – 37
vinylin – 18
aerosol – 10
quercetin – 4
three sodium ethylene
diamine tetra acetate – 1
For dental bleaching at home, the composition is:
carbamide peroxide – 15
glycerol – 46
vinylin – 22
aerosol – 12
quercetin – 4
three sodium ethylene
diamine tetra acetate – 1
The components of the two prescriptions are compatible with each other, and their quantitative and qualitative characteristics correspond to the concentrations described in the Pharmacopoeial Articles, the Temporary Pharmacopoeial Articles, the specifications and the instructions for use.
Unlike most of the bleaching agents, the above-mentioned agents have a pH of 6.8–7.1, which prevents the demineralization of the surface layer of enamel. The bleach was kept for 20 days in a light-proof package.
The
It should also be noted that carbamide СО(NH2)2 neutralizes acids (primarily milk) that produce microorganisms of the dental plaque. This process is due to the cleavage of the urea with the enzyme with the formation of carbon dioxide (CO2) and ammonia (NH3). Since NH3 has an alkaline environment, it neutralizes the acid. The presence of aerosil composition, which is silicon sorbent, prolongs the action of the active component – urea peroxide and its gradual removal from the therapeutic form. Vinylin is known for its anti-inflammatory, enveloping, and regenerating effect, which also reduces the irritant effect of urea peroxide on gum in the formulation. 3-natrium EDTA was introduced into the bleaching composition as a stabilizer that prevents catalytic decomposition of urea peroxide.
At the initial stage, the vestibular surface of the enamel of intact teeth treated by the method of home (14 teeth) and clinical (12 teeth) bleaching in patients aged 17–35 years old was removed by orthodontic indexes. The oral surface of the same experimental teeth served as control.
After the last bleaching procedure, the teeth to be prepared were purified by the end of the brush and washed with distilled water. Enamel was ground with a diamond bur at a rate of 2000 rpm. Overheating of solid tissues was prevented by periodic irrigation of tissues with distilled water and by drying them in a stream of air, as well as by intermittent loading of a rotating tool on a tooth. The sample of enamel was thoroughly triturated in an agate mortar and sieved through a sieve with a diameter of holes of 0.04 mm. To remove moisture and to prevent the chemical activity of potassium bromide (KBr) against hydroxyapatite, the powder was dried at 100–105 °C for 3 hours in a drying cabinet. A hard powder of enamel (3 mg) was mixed in an agate mortar with 300 mg of potassium bromide, the particle size of which was about 30 ... 40 microns. The obtained samples were the same mass, which made it possible to quantify the relative optical density of absorption bands of the
After obtaining the results, the parameters of each individual absorption band were analyzed and the concentration of the substance in the samples was determined. The analysis was based on the dependence of the intensity of absorption bands on the concentration of the test substance according to the Beer-Lambert-Bouguer formula (
at that
In order to process and analyze the results obtained, methods of variation statistics were used: the estimation of the difference between the frequency of occurrence of the characteristic in separate series of observations, the comparison of serial values and the mean square deviation. To assess the significance of the difference in the indices of the two populations obtained during the study, the degree of difference between their mean scores was determined using the Student’s t-test. The difference in statistical variables was considered significant at p<0.05. “Statistica for Windows" advanced analytics software, “Excel Statistica 7.0" spreadsheets (“Microsoft", USA) were used.
An analysis of the results of infrared spectra of intact enamel revealed the types of oscillations of valence grouping of organic and mineral components characteristic of enamel. In the range of 4000 ... 400 cm-1 (Fig.
565 cm-1 and 603 cm-1 – asymmetric deformation vibrations ν4 (PO43-)
872 cm-1 – non-expressed vibrations ν2 (СО32-)
960 cm-1 – complete metric valence fluctuations ν1 (PO43-)
1040 cm-1 and 1085 cm-1 – asymmetric valence fluctuations ν3 (PO43-)
1417 cm-1, 1457 cm-1 – asymmetric flat valence vibrations ν3 of СО32- ion
1540 cm-1 – Amide II valence oscillations (NH)
1650 cm-1 – Amide I and valence oscillations (C-O)
2849 cm-1 and 2920 cm-1 – valence fluctuations (C-H), (NH)
3430 cm-1 – oscillations of ОН-group of molecules of tightly bound water
In assessing the
The infrared spectrum of intact enamel.
The valence fluctuations of the amide groups of proteins are in the range of 2920–2860 cm-1, 2390–2370 cm-1, and 1700–1500 cm-1. The valence fluctuations of NH groups are the most important among them, which are designated as Amide II and the carbonyl strip Amid I. The valence vibrations in the region of 2920–2860 cm-1 belong to weakly evident valence fluctuations of CH- and NH-groups. Valid oscillations of Amide II, with a maximum at 1540 cm-1, are due to plane fluctuations accompanied by the rotation of the N-H bond. Therefore, the band Amide I, with a maximum at 1660 cm-1 (carbonyl), is characteristic of oscillations, in which the length of the C-O bond changes. The position of these bands is a function of conformation of the protein, in addition, they are connected with deformation oscillations of the bond O = C-N-H. The absorption band of Amide II band exceeds the absorption area of Amide I, probably due to the absence of collagen proteins in the enamel. The protein matrix of the enamel is represented by the three-dimensional protein-calcium-protein structure, the basis of which is glycosaminoglycans and Ca2+ ions. The mature, formed enamel almost does not contain proline and oxyproline (
The absorption bands of groups that have hydrogen atoms are located at the short-wave end of the fundamental frequency of oscillations of 4000–2000 cm-1 molecules. The greatest frequencies are characterized by the band of valence fluctuations of free hydroxide groups, which are in the region of 3650–3590 cm-1. The absorption band with a maximum at 3340 cm-1 relates to oscillations of strongly coupled water molecules (OH-) (
Thus, the mineral enamel matrix is mainly composed of insoluble forms of trisubstituted calcium phosphate Ca3 (PO4)2, which is part of the hydroxyapatite. In addition, valence oscillations of orthophosphates, characterized by different intensive bands, have been detected; For example, strips of weak intensity in the range of 1090–1030 cm-1 characterize soluble forms of phosphates (НРО42-). At the same time, the presence of carbonates has been noted with orthophosphates in crystalline hydroxyapatite (
According to the authors (
As evidenced by the results of the enamel study, after the application of the drug composition with 30% or 15% of CP (Figs
The infrared spectrum of enamel after treatment with bleaching preparations with different CP concentrations and performed remineralizing therapy.
Analysis of the mineral component of the enamel allowed determining that the parameters of the characteristic band with a maximum at 1040 cm-1 remain the largest. The decrease in the intensity of the valence fluctuations of the PO43- phosphate anion in the spectral region of 1090–1040 cm-1 was noted. No condensed phosphate groups were found that would be characterized by two types of bridge-links P-O-P with fluctuations of 750 cm-1 and 710 cm-1. In our opinion, their absence is due to the greater sensitivity of the bridge links of P-O-P to the influence of oral fluid, in contrast to the stable orthophosphate groups contained in the hydroxyapatite crystal.
In the oscillation region of the connections of the SO32- anion, the absorption bands were determined due to asymmetric flat valence vibrations ν3 with maxima at 1417 cm-1 and 1457 cm-1, as well as unexpressed valence fluctuations ν2 (880–875 cm-1). The parameters of these absorption bands are similar in the control group of samples.
Our obtained results indicate that the absorption band intensity of the hydroxide groups of the short-wave spectrum is reduced, as well as the bandwidths of the valence oscillations of the groups (CH- and NH-) with maxima at 2920 cm-1, 2870 cm-1, 2380 cm-1.
To determine the quantitative changes in the organic and mineral components of the enamel processed by 30% and 15% preparations of dental CP, we carried out the calculation of the optical densities of absorption bands of enamel. The analysis of the mineral enamel matrix has established the stability of the valence grouping parameters in enamel samples processed by different CP concentrations, as well as the lack of changes characterizing the destruction of the organic enamel matrix of the tooth in the Amide I and Amide II regions.
According to the results of the study, after the action of 30% CP, statistically significant changes (p < 0.01) of optical density occurred in the region of the band with a frequency of 3430 cm-1 (water, hydroxide groups), 2920 cm-1 (CH), 2380 cm-1 (НРО42-, СН, NH), as well as a tendency (р > 0.1), in relation to changes in the frequencies of Amide I (1650 cm-1) and Amide II (1540 cm-1).
In the group of enamel samples, a 30% carbamide peroxide preparation determined a statistically significant decrease of 2.17% of the optical density of the band with a frequency of 3430 cm-1 (p < 0.01). Such changes can be assumed to occur due to the ability of urea to form strong hydrogen bonds and to “drag" free water to “itself", that is, towards the surface of the enamel. The water balance of the enamel is restored due to the ability of the centripetal motion of the enamel liquor in the state of the pulp. Taking the above mentioned into consideration, subject to the bleaching of the vital teeth, it can be assumed that changes in the number of hydroxide groups of free and bound water are temporary and reversible.
When comparing the prolonged effect of a bleaching agent of lower concentration (15% CP), the character of the changes in the band of hydroxide groups with a frequency of 3430 cm-1 indicates that the tendency to enamel deamination is preserved (p < 0.05). Compared with the control, the optical density of this band decreased by 1.44%. The figures obtained are summarized in Table
The infrared spectrum of enamel after the action of bleaching preparations with different concentrations.
Indices of the optical density of absorption bands of enamel (M ± m), exposed to bleaching agents with different concentrations of urea peroxide.
Bandwidth absorption (cm -1) | Optical density of absorption bands of enamel (conditional units) | ||
---|---|---|---|
Control (n = 17) | The enamel exposed to the medications | ||
30% carbamide peroxide ( |
15% carbamide peroxide ( |
||
3430 | 0.138 ± 0.004 | 0.135 ± 0.021** | 0.136 ± 0.018** |
2920 | 0.062 ± 0.040 | 0.058 ± 0.06** | 0.061 ± 0.002* |
2870 | 0.041 ± 0.003 | 0.039 ± 0.003** | 0.04 ± 0.002* |
2380 | 0.006 ± 0.007 | 0.0078 ± 0.002** | 0.066 ± 0.060** |
1650 | 0.047 ± 0.006 | 0.046 ± 0.048*** | 0.046 ± 0.021*** |
1540 | 0.064 ± 0.012 | 0.063 ± 0.087*** | 0.063 ± 0.046*** |
1456 | 0.138 ± 0.002 | 0.138 ± 0.013*** | 0.138 ± 0.025*** |
1417 | 0.128 ± 0.002 | 0.128 ± 0.023*** | 0.128 ± 0.044*** |
1084 | 0.846 ± 0.012 | 0.845 ± 0.011*** | 0.845 ± 0.016*** |
1040 | 1.326 ± 0.015 | 1.323 ± 0.093** | 1.325 ± 0.017** |
960 | 0.255 ± 0.003 | 0.255 ± 0.043*** | 0.255 ± 0.062*** |
874 | 0.075 ± 0.003 | 0.077 ±0.012* | 0.077 ± 0.014* |
603 | 0.432 ± 0.033 | 0.427 ± 0.035** | 0.426 ± 0.073** |
566 | 0.437 ± 0.014 | 0.436 ± 0.023* | 0.436 ± 0.013* |
* – indicator of the likelihood of difference, compared with control at р < 0.05; ** – - р < 0.01; *** – - р > 0.1
The results indicate that teeth whitening with 15%
Valence oscillations of the amide groups of proteins are localized in the range of 2920–2860 cm-1, 2390–2370 cm-1, 1700–1500 cm-1. In the analysis of the oscillation frequency of 2920 cm-1 and 2870 cm-1, it should be noted that these absorption bands, in addition to the fluctuations of C-H groups, characterize the variations of the hydroxide groups bonded by hydrogen bonds, as well as the R- NH3+, aldehyde, and carboxyl groups. Carboxylic acids and aldehydes should be excluded from consideration since they do not have an absorption characteristic on the spectrum with a frequency of 1700 cm-1.
The analysis of the results of the infrared spectrum of the enamel processed by 30% of the
In addition, attention should be paid to the changes in the region of the spectrum of 2390–2370 cm-1, where the intensity band was reached at a maximum of 2380 cm-1 (НРО42-, C-H- and N-H-grouping). The optical density of this band after the application of 30% of the
To determine the state of the mineral component of bleached teeth, the analysis of the optical densities of absorption bands of orthophosphate-specific bands with maxima of 1085 cm-1, 1040 cm-1, 960 cm-1, 566 cm-1 and carbonates with maxima at 1456 cm-1 and 1417 cm-1, respectively was conducted. The frequencies in the range of 1000–1100 cm-1 are due to the fluctuation of the Н2РО4-, НРО42- and НРО43- ions, and at the frequency of 960 cm-1, the absorption of the РО43-group supplements the absorption of δ (С-Н). Accordingly, it is problematic to determine the changes in the valence oscillations inherent in the РО43- anion, or C-N groups.
After application of 30% or 15% of the CP preparation, no significant decrease in the optical density in the orthophosphate region with absorption band maxima at 1085 cm-1, 1040 cm-1, 960 cm-1 (p > 0.1) (Tab.
In the frequency region of 1456 cm-1 and 1417 cm-1, corresponding to the variation ν1 of the СО32- ion, and also δ C-H, no probable changes in optical absorption density were detected (p>0.1). In the range of 900–850 cm-1 spectrum, after the treatment of the teeth with 15% and 30%
The character of valence groups of bleached enamel after remineralization therapy is as follows. It should be noted that remineralization therapy has taken place since the fifth session of the use of bleaching substances. To do this, at the end of the teeth whitening session, the “Belagel Ca/P" gel preparation was applied on the cleaned and dried surface of the enamel, which was saturated with Ca and P ions for a prolonged time. The
The study of the spectrograms of the experimental samples did not reveal changes in the number of absorption bands and their localization in comparison with the control group, which indicates the absence of additional inclusions in the chemical structure of the enamel (Table
Indices of the optical density of absorption bands of enamel (M ±m), after treatment with bleaching agents with different CP concentrations and remineralizing therapy.
Frequency of absorption bands, (см –1) | Optical density of absorption bands of enamel (conditional units) | ||
---|---|---|---|
Control (n = 17) | The enamel exposed to the medications | ||
30% carbamide peroxide (CP), «Belagel Ca/P» (n = 12) | 15% carbamide peroxide (CP), «Belagel Ca/P» (n = 14) | ||
3430 | 0.138 ± 0.004 | 0.143 ± 0.005** | 0.142 ± 0.003** |
2920 | 0.062 ± 0.040 | 0.061 ± 0.005* | 0.06 ± 0.004** |
2870 | 0.041 ± 0.003 | 0.04 ± 0.024* | 0.04 ± 0.009* |
2380 | 0.006 ± 0.007 | 0.0076 ± 0.003** | 0.0075 ± 0.007** |
1650 | 0.047 ± 0.006 | 0.046 ± 0.072*** | 0.046 ± 0.061*** |
1540 | 0.064 ± 0.012 | 0.063 ± 0.058*** | 0.063 ± 0.092*** |
1456 | 0.138 ± 0.002 | 0.139 ± 0.043*** | 0.138 ± 0.074*** |
1417 | 0.128 ± 0.002 | 0.127 ± 0.013*** | 0.127 ± 0.042*** |
1084 | 0.846 ± 0.012 | 0.845 ± 0.01*** | 0.845 ± 0.013*** |
1040 | 1.326 ± 0.015 | 1.329 ± 0.014** | 1.328 ± 0.014** |
960 | 0.255 ± 0.003 | 0.254 ± 0.004*** | 0.255 ± 0.003*** |
874 | 0.075 ± 0.003 | 0.078 ± 0.003** | 0.079 ± 0.003** |
603 | 0.432 ± 0.033 | 0.429 ± 0.034** | 0.427 ± 0.038** |
566 | 0.437 ± 0.014 | 0.438 ± 0.037*** | 0.437 ± 0.052*** |
* – indicator of the likelihood of difference, compared with control at р < 0.05; ** – - р < 0.01; *** – - р > 0.1
Further analysis of the absorption strips of bleached enamel samples and the Belagel Ca/P was performed at 2920–2860 cm-1 (p < 0.01). The obtained results indicate that the use of remineralizing agent does not affect the process of destruction of the carbon bonds of CH- and NH-groups and the removal of the restructured organic component of the enamel (probably this process relates to the chromoform of the pigments) when bleaching the teeth. A probable reduction of the absorption band with a maximum at 603 cm-1 was determined, and an increase in the optical density index with a maximum at 874 cm-1 and 1040 cm-1, indicating a process of saturation of enamel with НРО42- ions (p < 0.05).
The use of remineralizing therapy leads to a possible increase in the optical density of the band with a maximum at 3430 cm-1 (p < 0.01) in the case of teeth whitening in clinical conditions by 5.79%, and at home – by 3.74% – according to indicators of bleached teeth.
Taking into account the obtained results, it can be assumed that the remineralization capacity of the oral fluid and the pulp, the state of hydration membranes, osmotic pressure of the enamel liquor is restored in the enamel due to the compensatory processes caused by the action of the Belagel Ca/P. Because of these activities, the processes of ion exchange are improved. Therefore, the use of remineralizing therapy in a clinical study of bleaching agents can lead to the disappearance of an unpleasant sensation during tooth whitening due to changes in the pulp.
Thus, the obtained results of the study indicate that the process of discoloration of teeth is accompanied by changes in carbon bonds of СН- and NH-groups, a decrease in the number of OH-groups and dissolved forms of phosphates. When applying medicinal compositions, in accordance with the elaborated methods of bleaching the vital teeth, no significant changes in the enamel are revealed, which would indicate the destruction of the hydroxyapatite crystals or its organic matrix. To seal the crystal lattice of enamel, the use of remineralizing therapy with drugs containing Ca and R ions is recommended.