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ORIGINAL RESEARCH
Year : 2021  |  Volume : 12  |  Issue : 1  |  Page : 8-14

Physical and Chemical Characterizations of Novel Bioactive Caries Detecting Solution


1 Department of Conservative Dentistry and Endodontics, Institute of Dental Sciences, Siksha ‘O’ Anusandhan (Deemed to be) University, Bhubaneswar, Odisha, India
2 Department of Physics and Semiconductor Research Laboratory, Siksha ‘O’ Anusandhan (Deemed to be) University, Odisha, Bhubaneswar, India
3 Department of Chemistry, Faculty of Engineering and Technology (ITER), Siksha ‘O’ Anusandhan (Deemed to be) University, Odisha, Bhubaneswar, India
4 Department of Conservative Dentistry and Endodontics, Sriram Chandra Bhanja Govt Dental College & Hospital, Cuttack, Odisha, India
5 Department of Oral Pathology and Microbiology, Institute of Dental Sciences, Siksha ‘O’ Anusandhan (Deemed to be) University, Odisha, Bhubaneswar, India

Date of Submission06-Jul-2020
Date of Decision22-Oct-2020
Date of Acceptance01-Dec-2020
Date of Web Publication2-Mar-2021

Correspondence Address:
BDS Shashirekha Govind
MDS (Prof & Head), Department of Conservative Dentistry and Endodontics, Institute of Dental Sciences, Siksha ‘O’ Anusandhan (Deemed to be) University, K-8, Kalinga Nagar, Ghatikia, Bhubaneswar, Odisha 751003
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/denthyp.denthyp_90_20

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  Abstract 


Introduction: The nature and progression of acute and chronic carious lesion are extremely variable on different tooth surfaces. Early detection of dental caries is challenging for clinicians and involves careful visual and tactile examination. Caries detection dyes and chemomechanical caries removal solutions guide the clinicians in the removal of infected dentin. This study aims to prepare and analyze the physical, chemical, and thermal characterizations of novel bioactive caries detecting dye solution (BCD) and its effectiveness in caries removal from the tooth structure. Materials and Methods: BCD is a combination of contrast agent (iobitridol), chitosan (CS), nanohydroxyapatite (nHAP), and coloring agent. It is synthesized, lyophilized, and subjected to Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD), and differential scanning calorimeter (DSC) analysis. Pilot study was conducted by applying BCD on two extracted carious teeth for evaluating caries removal efficiency under a microscope (10x). Scanning electron microscopy (SEM) image analysis was done to assess the percentage of dentinal tubules occlusion. Results: FTIR, XRD, and DSC analysis revealed that BCD has compatible interfacial bond between the components and is endothermic. Effective caries removal was seen under a microscope and SEM analysis revealed mean 77.66% of dentinal tubules occlusion. Conclusion: BCD is a stable solution without exothermic reaction, has caries identifying potential, and helps in caries removal. BCD is also bioactive in nature due to the presence of CS and nHAP as ingredients.

Keywords: Bioactive material, chitosan, contrast media, dental caries, nanohydroxyapatite


How to cite this article:
Govind S, Kamilla SK, Nanda B, Jena A, Mohanty N. Physical and Chemical Characterizations of Novel Bioactive Caries Detecting Solution. Dent Hypotheses 2021;12:8-14

How to cite this URL:
Govind S, Kamilla SK, Nanda B, Jena A, Mohanty N. Physical and Chemical Characterizations of Novel Bioactive Caries Detecting Solution. Dent Hypotheses [serial online] 2021 [cited 2021 Apr 11];12:8-14. Available from: http://www.dentalhypotheses.com/text.asp?2021/12/1/8/310538




  Introduction Top


Material science has a significant role in diagnosing and managing dental caries, which always has remained a challenge. Multifarious materials and methods have been implemented to face this challenge, including conventional techniques, such as visual inspection, radiographic method, and caries detecting dyes (traditional and fluorescent dyes), and advanced methods, such as quantitative light induced fluorescence, Diagnodent, and Calcivis. Extensive studies with caries dyes and chemomechanical caries removal methods have been considered minimally invasive, but they are time-consuming and compromise clinical outcomes.[1],[2] Dyes are developed to guide the clinician for the removal of the infected dentin. The use of natural dyes (blue from indigo, yellow from turmeric and saffron, red from safflower, lac, and madder) is uncommon as compared to synthetic dyes (acid red, Alexa Fluor 594, amino fluorescein, and lucifer yellow CH) in caries detection. There is scepticism that synthetic dyes are nonselective and results in the excessive excavation of sound dentin.[3]

Limited application of contrast media for diagnosis/identification of dental caries is verified in the literature.[4] Contrast media (ionic/nonionic) are used to enhance internal structures on imaging modalities and do not cause discoloration of the organs. Studies have suggested that contrast media like iobitridol [Xenetix 350, Guerbet (Roissy CDG, France)] is water-soluble, nonionic with low osmolarity, and does not react with membrane proteins of the cell wall.[5],[6],[7]

Chitosan (CS) has essential properties such as antimicrobial, anti-inflammatory, bioactive, biomimetic,[8] biocompatible,[9] promoting bone formation,[10] and biodegradability. Electrospun CS nanofibre mats are used for caries prevention[11],[12] and CS mixed with iopamidol (a nonionic contrast agent) is used for diagnosing circulatory disorders.[13] It also has wide applications in various biomedical and clinical fields.[14]

Nanohydroxyapatite (nHAP) [Ca5(PO4)3OH] is the main constituent of teeth and bones and has a hexagonal crystal unit cell. Its application includes drug delivery systems, orthopedics, enamel repair,[15],[16] filler for bone resorption, reduction of dentin hypersensitivity (DH), coating for prosthetic implants, and injectable bone cementation.[17],[18] The CS-nHAP composite material is biocompatible with enhanced bioactivity in tissue regeneration, proliferation of progenitor cells, and osteogenic differentiation of osteoblast-like cells.[18],[19],[20]

Thus, the present study aims to: a) prepare bioactive caries detecting dye solution (BCD), b) study the physical and chemical characterization of BCD by Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD) analysis, and differential scanning calorimeter (DSC) analysis, and c) carry out in-vitro evaluation of caries removal efficiency on extracted teeth and scanning electron microscopy (SEM) analysis for dentinal tubules occlusion.


  Materials and Methods Top


Synthesis of BCD: The study was approved by Siksha ‘O’ Anusandhan (Deemed to be) University/Institutional ethical committee, Odisha, India (Number: DMR/IMS.SH/SOA/180168) (IP India Patent application no: 201831039005).

Iobitridol [Xenetix 350 (nonionic; Guerbet, Roissy CDG, France)], 3% CS (I-chess, Mumbai, India), 15% nHAP (NanoXIM Care, Fluidinova, Portugal), and laccaic acid (Perma Colours & Chemicals Pvt. Ltd., West Bengal, India) were mixed to obtain a homogeneous BCD by using a magnetic stirrer at room temperature for 30 min. The mixture’s pH was determined with a digital pH meter (Eutech pH 700, Thermo Fisher Scientific, USA) and was maintained between 6.5 and 7.

Solidification (composite form) of BCD solution by Lyophilization procedure: This technique was based on the sublimation process (primary drying) followed by desorption (secondary drying). The samples were freeze-dried under vacuum without the chance for any biological growth or chemical reaction. Under low temperature and air pressure, the samples were heated, converting the water into a gaseous state (steam). The released vapor condenses in a cooling coil and undergoes desorption. The phases used were:
  1. Process of freezing: 10 mL of the BCD was frozen between −40°C to −45°C and the liquid phase converted to the ice-like solid phase.
  2. Sublimation (primary drying): The intramolecular water was sublimated; the water evaporated, captivated, and resolidified on cold condenser plates at −60°C to −70°C.
  3. Desorption (secondary drying): The obtained product was heated to a maximum temperature of +50°C, resulting in a freeze-dried solid product.


The obtained solid form of the BCD was subjected to FTIR, XRD, and DSC analysis.

FTIR: To analyze the different functional groups present in the composite, 50 mg of BCD powder was mixed with 5 mg of potassium bromide powder (binding agent), compressed to form a thin pallet, placed in a specimen holder, and transferred to the spectrophotometer for analysis. FTIR (Perkin, Elmer, Spectrum one, Texas, USA) was done using diffuse-reflection scanning mode over a wave number of 450-4000 cm−1 with a resolution of 4 cm−1 and analyzed by plotting the spectra transmittance versus wave number (cm−1).

XRD: The phase analysis of the sample was carried out by XRD (Model PW 1729, Philips, Holland) using 35 mA and 40 kV current. A monochromatic CuKα (target) radiation (λ = 1.5406 A°) with a step size of 0.04°, scan rate of 0.02°/s, and a scan range from 20° to 60° was used.

DSC: It evaluated BCD’s thermal behavior and determined the fraction of nHAP and its degree of crystalline behavior in the composite. Calibration of the DSC instrument (Model TA Q-20, TA Instruments, Bengaluru, India) was done using 10 mg of standard indium. Nitrogen was used as a nonreactive gas with 20 psi at a 50 mL/min flow rate through the DSC cell. 10 mg of lyophilized BCD composite material was placed in a T-zero hermetic aluminium pan. The pan was placed in a sealed holder with indention facing down and placed in the reference slot. The experimental model was as follows: modulation amplitude was set at 0.19°C and the underlying heat rate was maintained at 2°C/min with a modulation period of 40 s. At 25°C, the process consisted of 20 min isothermal period to allow equilibration of the sample, and then the heating was maintained between −80°C to 400°C.

SEM: Pilot study was conducted on two carious extracted molar teeth (unknown history) as per university guidelines. Teeth were disinfected with 0.2% thymol solution and stored in artificial saliva (wet mouth, ICPA Health product Ltd, Mumbai Maharashtra, India). The carious teeth were mounted in modelling wax till cementoenamel junction level. 0.5 mL of the BCD was scrubbed for 40 s with a microbrush on the carious surface and radiovisuography (Schick CDR International, X-Mind DC, Acteon, Italy) was taken. Mechanical caries excavation was done with an oval shape spoon excavator (1Exc31L: GDC Fine Crafted Dental Pvt. Ltd., Hoshiarpur, India) using a dental operating microscope (Model: Alpha Slim 6, Seiler Instrument Inc., St. Louis, USA) at 10× magnification [Figure 1]a–g. The excavated teeth surfaces were evaluated for color (visually) and hardness (tactile sensation with probe). A diamond disc (NTI® Flex D345-190 Kerr, California, USA) was used to section the teeth mesiodistally [Figure 1]:h,i. Dentin samples (3 × 3 × 2 mm) comprising peripheral and deep occlusal surface samples (four each) from the excavated region and four samples from noncarious teeth regions were used to visualize dentinal tubules occlusion. Sections were thoroughly washed in distilled water, dried, gold sputtered to form a thin layer by sputter coating machine (Q150R ES, GS Quorum Technologies Ltd, Laughton, England), and photomicrographic images were taken using SEM (Merlin Compact, Carl Zeiss Microscopy GmbH, Munich, Germany) from between 1500× to 2000× at 5 kV. The percentage of occluded dentinal tubules was calculated by dividing the total number of occluded tubules (full and partial) by the total number of tubules in the photomicrograph and multiplied by 100.[21]
Figure 1 Illustrates the application of BCD solution on extracted teeth: (a) Preoperative carious tooth, (b) BCD applied, (c) preoperative radiographic image of caries, (d) radiographic image showing radiopaque extension of BCD, (e) radiograph after caries excavation, (f) soft caries removal by spoon excavator, (g) effective caries removal, (h,i) mesio-distal sectioned tooth surface with black arrow indicating no residual BCD and red arrow indicating the noncarious coronal dentin surface; (i) Clean tooth sections (16× magnification) prior to SEM analysis. BCD, bioactive caries detecting dye solution; SEM, scanning electron microscopy.

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  Results Top


FTIR analysis: The infrared spectra of nHAP-CS-iobitridol are shown in [Figure 2]. The intense band near 3365–3253 cm−1 confirms the presence of N-H and O-H stretching and intramolecular hydrogen bonding. The spectra show the C-H symmetric and asymmetric stretching band near 2928 and 2875 cm−1 respectively, due to the presence of polysaccharides. The band near 1642 cm−1 endorses the occurrence of C═O stretching of amide I (residual N-acetyl groups), and the band near 1557 cm−1 corresponds to the N-H bending of amide II. A band near 1399 cm−1 is mainly due to the presence of CH3 symmetrical deformations. The bending vibration signal at 1268 cm−1 confirms the presence of hydroxyl group in CS. Bands near 1091 and 1029 cm−1 are attributed to C-O stretching. The typical signals observed around 1029, 602, and 563 cm−1 are due to nHAP, attributed to P-OH stretching and corresponding phosphate (PO43–). The prominent peak of nHAP found near 633 cm−1 denotes the OH end group with a lower steric impediment of the structure. From this, it is understood that the composite of nHAP/CS is stable.
Figure 2 Illustration of peaks for BCD material by FTIR. BCD, bioactive caries detecting dye solution; FTIR, Fourier transformed infrared spectroscopy.

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XRD analysis: As illustrated in [Figure 3], XRD revealed the structural information about the composite’s individual components. The diffraction peaks at 14°, having a (020) plane, confirm that CS exhibits partial crystallinity and orthorhombic structure.[11],[22] After the modification of CS with nHAP, the crystallinity gradually decreases. nHAP shows the diffraction peak near 25.6°, which is shifted to higher 2θ values (28.52° and 31.94°) due to nHAP/CS composite formation. The shift is because of the compression from the contracting polymer matrix through interfacial bonding. The peak shifting and decrease in the crystallinity indicate the presence of bonding between the nHAP particles and the CS polymer matrix. The diffraction peak 2θ of nHAP is depicted at 31.94° having a plane (300) and is weakened after the formation of composite, which indicates the participation of nHAP in bonding with CS.
Figure 3 Illustration of peaks for BCD material by XRD. BCD, bioactive caries detecting dye solution; XRD, X-ray diffraction.

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DSC analysis: A weak endothermic peak occurred between 50°C and 150°C with intact water desorption at 100°C. Thermo degradation of CS having increased exothermic peak is supplemented with the inclusion of nHAP in the composite compound. The DSC curve temperature peaks appear for CS but shifted to a lower temperature for nHAP/CS composite [Figure 4].
Figure 4 Illustration of peaks for BCD material by DSC. BCD, bioactive caries detecting dye solution; DSC, differential scanning calorimeter.

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SEM analysis: The excavated surface was yellow/light yellow [Figure 1]g, and the hard surface on probing was indicative of soft carious dentin removal. Radiographic images [Figure 1]d reveal the extension of BCD, indicating caries extension. The mean percentage of dentinal tubules occlusion for excavated peripheral coronal surface and near pulpal surface area was 74.4 and 79.07, respectively [Figure 5]a,c,e. SEM analysis revealed mean 77.66% of dentinal tubules occlusion. Patent open dentinal tubules were seen in noncarious coronal dentin surfaces [Figure 5]b,d,f.
Figure 5 Photomicrographs of dentinal tubule occlusion by nHAP crystals: (a) Peripheral excavated surface, (c,e) deep excavated surface near to pulp area, and (b,d,f) open dentinal tubules on noncarious coronal tooth surface.

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  Discussion Top


In the current study, BCD was prepared as a homogeneous solution and lyophilized as solid samples are convenient for characterization analysis. FTIR analysis of BCD showed iobitridol, CS, and nHAP in 3365–3253 cm−1 region corresponding to N-H and O-H stretching and has intramolecular hydrogen bonds. The hydrogen bonds between the molecules is covalently bonded to highly electronegative elements (O, N, F), indicating that the molecules are soluble in water (polar solvent). This signifies that remnants of BCD can be flushed entirely with water.

XRD analysis revealed gradual decrease in crystallinity of CS, and the formation of nHAP/CS composite endorses that diffraction peak is shifted to higher 2θ values (28.52° and 31.94°). The shift and decrease in crystallinity of nHAP are weakened, and peak at 31.94° with plane (300) is obtained due to the formation of the composite. This may be correlated with the structural strength as there is chemical surface modifications and improved interfacial bonding between the materials resulting in a stable material.[17],[20] DSC curve shows the shift towards lower temperature, indicating that BCD is endothermic.

After application of the BCD, caries removal effectiveness was assessed under 10× magnification based on the color interpretation and tactile sensation of the hard dentin according to the parameters given by Hosoya et al.[23] The carious enamel-dentin consists of demineralized and degraded organic and inorganic components within the bacterial zone. Carious dentin has low pH, reduced permeability in the translucent zone due to caries crystals,[24] has dominant lactic acid bacteria, and bigger crystal size. In the X-ray spectrum, sound dentin showed more amorphous-like crystals.[25] At the surface of the carious dentin, the collagen’s cross-linkage showed virtual disappearance, indicating irreversible denaturation of the collagen.[25] However, BCD application on the carious surface with microbrush and scrubbing for 40 s helped in softening the degraded collagen and provided guided and gentle excavation with a spoon excavator. Active ingredient 3% CS (molecular weight 161.16 g/mol) is a good chelating agent with strong ability to bond with proteins[26] and an effective antimicrobial agent.[9] As pH decreases, the positive hydrogen ions from the acid bind to the negative hydroxyl and phosphate ions from the enamel, indicating mineral loss.[8],[27] This process can be inhibited by the application of BCD (pH 6.5–7), which contains protonated CS wherein the amino group helps capture the positive hydrogen ions from the acid, forming positive protective layer, and prevents binding of hydrogen ions to the mineral surface.[26]

Radiopaque extension of BCD [Figure 1]d is due to the presence of iobitridol: a nonionic, high radio-opacity, low toxic, and hydrophobic contrast agent.[5],[6] Well-appreciated radiolucency is seen after caries excavation [Figure 1]e. Studies have shown that iobitridol is clinically safe, and warmth sensation is commonly reported,[5],[6] but in BCD the exothermic property of iobitridol did not impact the solution. For detection of the caries lesion, bitewing radiography is considered a reliable method and has sensitivity and specificity of 0.4 and 0.9, respectively.[28] By application of BCD, the radiographic accuracy level for caries detection might be increased.

The density and diameter of the dentinal tubules increase from dentinoenamel junction [approximately 1% of the total surface area (15,000/ mm2)] to the pulp region [22% (65,000/mm2)].[29] Noncarious teeth shows increased dentin permeability with highest values for molars and lowest for premolars.[24],[30],[31] In the present study, SEM shows the occlusion of the dentinal tubules by nHAP crystals due to its easy dispersion from BCD during the scrubbing action, which helps in diffusion of nHAP into the dentinal tubules. In the peripheral excavated surface, majority of tubules were completely occluded [Figure 5]a. In the deep excavated surface, an agglomerated area and maximum partially occluded dentinal tubules were seen [Figure 5]c,e. The particle size of 15% nHAP crystals in BCD was <50 nm and molecular weight was 1004.6 g/mol. Previous studies have concluded that hydroxyapatite reduces dentin permeability in the absence of saliva [30] and is effective in reducing DH.[32] As nHAP is a component of BCD, it may have the potential to reduce dentin permeability.

BCD also has a natural dye; laccaic acid (food color) which provides green color to the solution and aid in the solution’s visual extension when applied on the carious tooth surface. After caries excavation, the sectioned tooth surface was devoid of any residual dye [Figure 1]:h,i as BCD can be washed off easily with water. BCD is physically stable without interaction with the environment, is endothermic, and preserves sound dentin. Future studies can be conducted to know the effect of BCD on the bond strength of the restorative materials and to determine its entireness for bacterial elimination clinically.


  Conclusion Top


The current work focused on chemical evidence for the efficacy of BCD on the carious dentin. BCD is a stable product that can be a good choice for diagnosing and removing dental caries and needs further optimization under clinically relevant conditions.

Acknowledgements

We acknowledge Dr Abhishek Pal, Assistant Professor, Gitam School of Pharmacy, Gitam Deemed to be University, Hyderabad for his support in conducting the experiments.

Ethics

The experiments carried after approval from Siksha ‘O’ Anusandhan (Deemed to be) University/Institutional ethical committee, Odisha India (number: DMR/IMS.SH/SOA/180168)

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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