Effect of electrophoresis on correlation between mineral content and enamel micro-hardness: a study in New Zealand rabbits

Introduction

Enamel is the toughest and most rigid substance found in the human body [1]. It is subject to various types of stresses from different directions serving as the protective outer layer of teeth. Its integrity is vital for maintaining oral health, and preventing caries and other dental diseases [2].
Globally, dental de-mineralization is common, and contemporary dentistry strives to manage non-cavitated white spot lesions (WSLs), prevent disease advancement, and preserve the structural strength of tooth material [3]. Re-mineralization of hard dental tissues is characterized as the mechanism by which calcium and phosphate ions from an external origin are provided to induce ion deposition into de-mineralized enamel [4, 5]. Lately, innovative techniques utilizing bio-mimetic mineralization without cells have demonstrated the ability to regene­rate tissues resembling enamel or dentin in laboratory settings. This advancement introduces the possibility of treating tooth imperfections using a self-repairing mechanism, representing far more favorable approach in dentistry compared with existing methods [6].
Substitutes for fluoride, such as nano-hydroxyapatite (n-HAP), casein phospho-peptide amorphous calcium phosphate (CPP-ACP), and bio-active glass (BAG) have been suggested for their re-mineralization proper­ties [6, 7]. CPP-ACP nano-complex, a phosphorylated casein peptide with multiple properties, helps sustain elevated levels of calcium and phosphate ions within the solution. Acting as a store of calcium-phosphate, CPP-ACP facilitates enhancing re-mineralization and reducing enamel de-mineralization [8, 9]. N-HAP, a highly bio-active and bio-compatible substance, has been increasingly embraced in the fields of dentistry and medicine. The utilization of nano-hydroxyapatite for mimicking the restoration of compromised enamel has gathered significant interest in contemporary dental studies due to its resemblances in terms of chemistry and structure with enamel minerals [10].
Bio-active glass is among the suggested agents for re-mineralization, and can be applied in the process of regenerating the tooth’s structure [11]. The main component in BAG binds to the tooth surface upon application, while in contact with saliva or other fluids, it initiates the process of re-mineralizing tooth enamel by providing silica, calcium, phosphorous, and sodium ions to the structure of the tooth [12]. It is important to mention that the speed of solid formation increases, potentially reducing the efficiency of these substances [13]. Ions have the capability to travel faster across a gel or liquid solution when undergoing electrophoresis, especially in ionophoresis. Additionally, it was asserted that electrophoresis, with the aid of an electric field and a minimal voltage of electrical current, facilitates the migration of ions along a specific one-dimensional route [14].
The hardness at a microscopic level of tooth enamel, which refers to how well the enamel can withstand being deformed and indented when subjected to a specific force, serves as a vital measure of its strength and general excellence as a dental substance [15]. In vitro, the charac­teristics of enamel caries lesions based on depth can be analyzed through X-ray attenuation (mineral content profiles) and mechanical properties (hardness profiles). Even though there are limited comparative data between micro-radiography and micro-hardness measurements, a connection has been observed [16].
Understanding the relationship between micro-hardness and mineral composition of enamel is crucial for dental experts when developing successful treatment plans and predicting the durability of dental repairs [15]. In a clinical setting, it is necessary to comprehend this connection. The impact of different substances on enamel toughness has been explored, but the link between mineral content of sound enamel and its micro-hardness is still unclear [15].

Objectives

The null hypothesis of the study was that there is a strong connection between calcium-to-phosphorus (Ca : P) ratio and micro-hardness, and electrophoresis technique has a positive effect on the correlation between Ca : P ratio and enamel micro-hardness.

Material and methods

The following materials were used in the present study: 1) CPP-ACP re-mineralizing agent in the form of tooth mousse (GC Tooth Mousse, batch number: 210408C, lot No.: 04008181GC, International, Itabashi-Ku, Tokyo, Japan); 2) n-HAP re-mineralizing agent in the form of toothpaste (Apagard Royal, batch number: 120-82-4101, Sangi, Co., Ltd., Japan); 3) BAG re-minera­lizing agent in the form of toothpaste (Biomin C, batch number: PMTB0039, BioMin Technologies Ltd., Bielefeld, Germany); 4) de-mineralizing agent in the form of 37% phosphoric acid was used for enamel de-mine­ralization (N-Etch, batch number: Z203PNM, Ivoclar, Vivadent, AG, Switzerland).

Sample size

ANOVA test was utilized to compare sub-groups in examining the impact of two distinct re-mineralization methods with 3 varied materials at 2 monitoring time points, based on a study conducted by Zhang et al. [17]. Around seven teeth in every sub-group were deemed satisfactory for identifying the impact size of 0.51 with a power of 0.8, employing a two-sided hypothesis test and a significance level of 0.05 for data analysis.

Teeth grouping

According to re-mineralizing agents used in the study, 84 teeth were randomly divided into two main, equally divided groups (n = 42) for electrophoresis and traditional methods. The teeth were further categorized into three equal sub-groups (n = 14), based on re-mine­ralizing agent utilized: sub-group N received n-HAP, sub-group C received CPP-ACP, and in sub-group B, BAG was applied. Each sub-group was then split into two equal parts (n = 7) based on the duration of re-mineralization. Traditional re-mineralization sub-groups were categorized into 2-week and 5-week periods, while electrophoresis re-mineralization groups were divided into 3-hour and 5-hour durations.

Intervention

Male, four-month-old New Zealand rabbits, weighing around 2 kg were put under anesthesia through a shot administered in the quadriceps femoral muscle via intra-muscular injection of 3.3 cm Xyla-ject solution, with a strength of 30 mg/kg. To keep the rabbits under control, an extra dosage of 10 mg/kg was given when needed during the experiment [18]. The study was approved by the Ethical Committee of the Faculty of Dental Medicine, (Boys-Cairo), Al-Azhar University (approval number: 120192/3/32). All animal testing methods followed the guidelines of the Canadian Council on Animal Care and aligned with the principles of the three ‘R’ in animal ethics, i.e., replacement, reduction, and refinement [19]. Additionally, the ARRIVE principles were followed, and no deaths occurred throughout the research phases.

Sample preparation

The upper front teeth underwent acid etching with 37% phosphoric acid (Ivoclar) for 60 seconds, to create a chalky white look resembling WSLs of tooth decay, followed by a thorough rinse with a significant quantity of deionized water [20].

Animal coding for re-mineralization techniques

Identification of animals with coding was carried out randomly to differentiate them while applying mate­rials. The process involved marking the inner ears of every rabbit with a permanent marker, using black color for n-HAP, red for CPP-ACP, and green for BAG.

Surface treatment

All re-mineralizing agents’ commercial products featured pastes as part of their contributions. The conventional method involved using a micro-brush on the labial surface of de-mineralized enamel specimens. Oral-B soft toothbrushes (Procter and Gamble Co., Cincinnati, Ohio, USA) were employed for brushing procedures in all groups. Gentle pressure was applied three times daily at eight-hour intervals using pure toothpaste (around 1 g) for one minute. Samples were then rinsed with deionized water for 15 seconds. After each brushing session, the treatment process was carried out for 2 and 5 weeks [21, 22]. For electrophoresis, a custom mold was designed featuring entry port on the outer surface. During electrophoresis, a setup employing two electrodes was utilized, connecting the anode to the skin of the rabbit, while the cathode was placed inside a specifically crafted mold containing CPP-ACP, n-HAP, or BAG. Electric flow was activated for a duration of 3 to 5 hours. Subsequent to each re-mineralization process, the upper incisors were removed [17].

Evaluation technique

Assessment of chemical composition (EDX results) – Ca/P minerals wt. %

Surface assessment was performed on test samples to determine the ratios, existence, and non-existence of minerals, especially calcium, phosphorus, and fluo­ride, using a scanning electron microscope (SEM) in combination with an energy-dispersive X-ray (SEM/EDX) microscope operating at 10 kV. It was outfitted with a detector and XP3 pulse processor (Oxford instruments X-ray micro-analysis, Oxford Super ATW). The samples underwent exposure to the electron beam at a take-off angle of 35°. EDX spectrum images were captured using the SPEC Vision integrated acquisition system. Assessment of surface micro-hardness A custom-made cylindrical plastic mold was fabricated with a 30 mm inner diameter and a 15 mm height [23]. Surface micro-hardness was assessed by utilizing a digital display Vickers micro-hardness tester (Model HVS-50, Vickers diamond indenter, 20× objective lens, Laizhou Huayin Testing Instrument Co., Ltd., China), and the specimens were tested under a 50 g load for a duration of 10 seconds. Three indentations were strategically positioned on each specimen’s surface, forming a circle with a minimum distance of 0.5 mm between them. Micro-hardness was determined using the following equation:
HV = 1.854 x P/d2,
where HV is the Vickers hardness measured in
kgf/mm2, P is the load in kgf, and d is the measurement of diagonals in millimeters.

Statistical analysis

Numerical data underwent assessment for normality through examination of data distribution and application of normality tests (Kolmogorov-Smirnov and Shapiro-Wilk tests). All data exhibited normal (parametric) distribution. Mean and standard deviation (SD) values were applied to present the data. Three-way ANOVA test was employed to investigate the impact of re-minera­lization technique, time, material, and their interplay on average hardness of Ca : P. Bonferroni’s post-hoc ana­lysis was employed for pair-wise comparisons in cases where ANOVA test yielded significant results. Pearson’s correlation was also utilized. A coefficient was used to establish the correlation among Ca : P, while the significance level was defined as p ≤ 0.05, focusing on the ratio and hardness. Data analysis was conducted with IBM SPSS Statistics for Windows, version 23.0 (IBM Corp., Armonk, NY, USA).

Results

Assessment of chemical composition (EDX results) – Ca/P minerals wt. %

Regardless of re-mineralization technique and time, there was statistically significant difference between Ca : P ratio of different materials. Pair-wise comparisons between the materials revealed that there was no statistically significant difference between CPP-ACP and BAG, as both showed statistically significantly lower mean Ca : P ratio than N-HAP (Table 1 and Figure 1).

Assessment of surface micro-hardness

Regardless of re-mineralization technique and time, there was statistically significant difference between hardness of different materials. Pair-wise comparisons between the materials revealed that there was no statistically significant difference between n-HAP and CPP-ACP, as both showed statistically significantly higher mean hardness than BAG (Table 2 and Figure 2).

Correlation between Ca : P ratio and hardness

There was no statistically significant correlation between Ca : P ratio and hardness using both re-mineralization techniques and all materials (Table 3 and Figure 3).

Discussion

WSLs are the initial visible signs of the onset of tooth decay, leading to a formation of complete cavity in the long term [17]. Due to their electrical charge, the crystals within the de-mineralized enamel have the ability to attract calcium and phosphate ions from the re-mineralization solution efficiently, thereby lowering surface energy levels [20]. Until now, the standard method for addressing teeth with cavities was to extract decayed tissues and substitute them with a restorative substance. Presently, a contemporary fluoride therapy has shown effectiveness in re-mineralizing white spot imperfections. However, this treatment is successful within an initial 10 to 30 µm of the spot, prompting the need for new therapies enabling re-mineralization of deeper regions [22].
Enamel is a delicate and non-uniform micro-structure, with a tendency to fracture during evaluating micro-hardness. In comparison with other hardness tests, Vickers hardness is quick, cost-effective, reliable, and does not cause damage [23, 24].
EDX analysis combined with SEM are commonly used techniques for investigating different solid samples. These methods help detecting minute morphological characteristics at micron and sub-micron scales as well as facilitate understanding of the chemical composition by determining the concentration percentages of elements present in samples.
The choice of these methods was based on their non-destructive nature and preservation of the sample’s volume during the X-ray generation process, enabling multiple analyses of the same sample [25].
Influential factors affecting micro-hardness of enamel encompass the level of mineralization, existence of organic elements, and external factors, such as pH and exposure to fluoride. Furthermore, fluctuations in micro-hardness may be associated with dietary factors and the occurrence of tooth decay, where de-mineralization mechanisms gradually diminish the hardness of ena­mel [15].
Mineral composition found in dental enamel is crucial in defining its strength and functionality. Hydroxyapatite stands as the main mineral element in enamel, forming a crystal structure from calcium and phosphate ions. This key mineral composition constitutes about 95% of enamel’s total weight, contributing to its distinct hardness and transparency. The connection between mineral levels and the structure of enamel is complex; elevated levels of hydroxyapatite result in a denser and well-arranged enamel framework, ultimately improving its mechanical characteristics [26]. For this purpose, in studies analyzing the mineral composition of tooth enamel, different methodologies, such as X-ray diffraction and SEM, were utilized. SEM offers detailed visuals of the enamel’s surface, showcasing the structural aspects impacting its functionality. Recent findings sug­gest that changes in mineral content are influenced by factors, such as diet and environment, which play a crucial role in shaping the enamel structure and determining its hardness [27]. Several studies have explored the association between micro-hardness and mineral composition in dental enamel, providing valuable insights into their mutual relationship. Moreover, studies have shown that enamel with higher mineral levels typically displays greater micro-hardness, supporting the idea that mineral density plays a significant role in the enamel’s ability to withstand wear and pressure [15, 28]. Furthermore, the implications of this relationship extend to dental care and treatment approaches, comprehending how mineral content and hardness interaction can contribute to preventive efforts against enamel erosion and guide restorative procedures [15]. This knowledge not only assists in preserving natural teeth, but also enhances the durability of dental treatments, leading to improved patient outcomes.
Regarding the correlation between Ca : P ratio and surface hardness, there was no statistically significant correlation found using both re-mineralization techniques and all materials, as shown in Table 3. This was in agreement with Herkströter et al. in 1989 [29] and Buchalla et al. in 2008 [16], who reported very weak or no direct relation between hardness and mineral content. This is in disagreement with Kielbassa et al. in 1999 [30], who found a correlation in the straight line between KHN and mineral composition.
Regarding re-mineralization periods, the results of the current study revealed a clinical difference found between 2 weeks and 5 weeks durations. Regardless of the applied surface treatment, week five provided the highest mean value in all tested groups. This may be related to the increasing contact time with the corresponding re-mineralizing agent, leading to the formation of an outer mineral-rich layer in the enamel, which consequently increases the percentage of mineral and in turn, it would be more resistant to acid attacks [31].
Electrophoresis can effectively accelerate the re-mineralization kinetic of re-mineralizing agents, and strengthen its re-hardening effects on de-mineralized enamel [32], but it does not have a positive effect on correlation between mineral content and micro-hardness.
In summary, the connection between micro-hardness and mineral composition in tooth enamel is a vital area of investigation in dentistry, with considerable impacts on oral well-being. A comprehensive understanding of the factors affecting the combination of micro-hardness data and knowledge about the mineral composition of enamel, enables dental professionals to customize preventive and restorative treatments more efficiently. As research progresses in this area, it is crucial to acknowledge the significance of preserving enamel integrity by promoting sufficient mineralization, thereby strengthening its hardness and resistance against decay. Future research studies should concentrate on investigating novel methods to enhance mineral levels in the enamel, ultimately ensuring the durability of dental health and effectiveness of restorative materials. By linking micro-hardness with mineral content, we can cultivate a deeper comprehension of the enamel’s role in dental well-being and contribute to the enhancement of preventive and therapeutic approaches in dentistry.

Conclusions

Under the circumstances of the study and limitation of the materials used, it may be concluded that the effects of re-mineralizing agents can be accelerated by electrophoresis. However, the present findings indicate that this method does not significantly influence the correlation between mineral content and micro-hardness. The null hypothesis of the study was rejected. Further research is necessary to explore the impact of varying electrical voltage and treatment duration on the correlation between mineral content and micro-hardness in electrophoretic re-mineralization.

Disclosures

1. Institutional review board statement: This study was conducted in accordance with all provisions in animal subjects, with committee guidelines and policies of the Faculty of Dental Medicine (Boys-Cairo), Al-Azhar University, Egypt. The approval code for this study is: 120192/3/32.
2. Assistance with the article: None.
3. Financial support and sponsorship: None.
4. Conflicts of interest: The authors declare no potential conflicts of interest concerning the research, authorship, and/or publication of this article.

References