ORIGINAL RESEARCH

https://doi.org/10.5005/jp-journals-10015-2277
World Journal of Dentistry
Volume 14 | Issue 7 | Year 2023

Evaluation of Quantum in Human Tooth Remineralization and Microhardness Potential with Two Types of Red Algae: An In Vitro Comparative Study

Yadav Chakravarthy¹, Vyapaka Pallavi², Aishwarya Santosh³, Vanita D Revankar⁴, Mohammed N Asmee⁵, Subhashini Manoharan⁶

^1–6Department of Conservative Dentistry and Endodontics, Vinayaka Mission’s Sankarachariyar Dental College, Salem, Tamil Nadu, India

Corresponding Author: Aishwarya Santosh, Department of Conservative Dentistry and Endodontics, Vinayaka Mission’s Sankarachariyar Dental College, Salem, Tamil Nadu, India, e-mail: aishwaryasantosh11@gmail.com

Received on: 01 June 2023; Accepted on: 03 July 2023; Published on: 31 August 2023

ABSTRACT

Aim: The study aimed to investigate and compare the remineralization potential of Lithothamnion calcareum and Lithothamnion superpositum on tooth enamel with commercially available casein phosphopeptide-amorphous calcium phosphate fluoride (CPP-ACPF).

Materials and methods: A total of 30 human third molars indicated for extraction was chosen for this study. Carious lesions depicting the early stages of tooth subsurface enamel lesions were produced by immersing tooth samples in a demineralization bath for 72 hours. The samples were divided into three groups of 10 each. Group I (control): CPP-ACPF, group II: Lithothamnion calcareum, and group III: Lithothamnion superpositum. The teeth were immersed in their respective solutions and the samples were then kept in a thermobath for 21 hours for 7 days following which they were then tested by Vickers microhardness test and X-ray fluorescence spectroscopy.

Results: Group II—Lithothamnion calcareum, exhibited a better remineralization potential of (224.35) than group III—Lithothamnion superpositum (215.64). Nonetheless, the commercial agent, a group I (CPP-ACPF), displayed the most efficiency in remineralizing tooth enamel.

Conclusion: Among the alternative potential remineralizing samples tested, group II—Lithothamnion calcareum, exhibited maximum remineralization potential value followed by other algae but was not to the extent of the commercially available CPP-ACPF. The results of this study with regard to the alternative tooth-enamel remineralizing samples tested, were promising.

Clinical significance: The alternative, a tooth-enamel remineralizing agent will serve as a natural and economical option for all sections of society, especially in rural India.

How to cite this article: Chakravarthy Y, Pallavi V, Santosh A, et al. Evaluation of Quantum in Human Tooth Remineralization and Microhardness Potential with Two Types of Red Algae: An In Vitro Comparative Study. World J Dent 2023;14(7):629–633.

Source of support: Nil

Conflict of interest: None

Keywords: Algae, Casein phosphopeptide-amorphous calcium phosphate fluoride, Demineralization, Lithothamnion calcareum, Lithothamnion superpositum, Remineralization, Vickers microhardness test, X-ray fluorescence spectroscopy

INTRODUCTION

Dental caries is one of the oldest and most common infections in humans. It is a multifactorial disease that is noncommunicable and biofilm-mediated which results in loss of minerals from tooth structure.¹^,² This is the outcome of a complicated association between plaque-adherent bacteria and fermentable carbohydrates, which results in demineralization of the enamel. Initially a “white spot lesion” is formed which eventually progresses to form cavitation. The white spot lesion if treated in its initial stages is reversible.³

In recent years, the dental practice has shifted toward minimally invasive dentistry which follows a more conservative technique that mainly emphasizes early caries detection, remineralization of dental surfaces, and maintenance of tooth structure surrounding the lesion. The cariostatic potential of fluoride has been established as the key contributor to the reduction of tooth caries. Despite its extraordinary potential to slow the progression of dental caries, it does not entirely remove it. Furthermore, an increase in fluoride concentration might have a negative impact on teeth. While conventional fluoride-based remineralization is the backbone of dental caries control, individuals and populations with a high caries risk typically require supplementary remineralizing agents to boost fluoride’s efficacy.⁴

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), is a commercial remineralizing agent which is produced from the milk protein casein. CPP-ACP is a source of bioavailable calcium and phosphate that aids in remineralization. Later, fluoride (0. 09%) was added to CPP-ACP, resulting in casein phosphopeptide-amorphous calcium phosphate fluoride (CPP-ACPF) paste. CPP-ACPF has a greater remineralization capability than CPP-ACP. However, these commercial remineralizing agents have certain disadvantages such as; short duration of action, providing only superficial remineralization, causing gastric irritation, allergies and are not viable for the low-income population.⁵

Over the years, various natural alternatives have been preferred such as corals, mollusks, egg shells, Moringa oleifera, fish, algae etc. Algae are aquatic, oxygen-evolving, photosynthetic organisms that are single-celled, colonial, composed of filaments, or made of simple tissues. There are an estimated 10–50 million species of algae.⁶

Red marine algae is a coralline alga belonging to the Hapalidiaceae family and Lithothamnion genus. The two species Lithothamnion calcareum and Lithothamnion superpositum were extensively studied. They thrive in cold Atlantic waters whose algae fronds collect minerals from seawater over their lifetime which eventually fall off. Minerals extracted from them include 12% calcium, 1% magnesium, and 72 additional trace minerals essential for bone health.⁶^,⁷ They possess high concentrations of calcium and other bone-forming elements. Literature has documented that these supplements have the ability to affect osteoblast proliferation and mineralization.⁶^,⁸ They render an alternative remineralizing agent that is socio-economically viable for all sections of society, especially the rural population. Therefore, it is important to evaluate their remineralization potential, and hence, the present in vitro comparative study aimed to investigate and compare the remineralization potential of Lithothamnion calcareum and Lithothamnion superpositum on tooth enamel with commercially available CPP-ACPF.

MATERIALS AND METHODOLOGY

This study was done in a private lab in Salem (Alpha Omega labs) from January to March 2022. This study was reviewed and approved by the Institutional Research Committee (IRC/20721/S/3).

The algae were obtained in their raw form from the RK Algae Project Center, Rameswaram, Tamil Nadu, India.

Sample Preparation⁹

Thirty human-impacted third molars which were extracted were selected for this study. After removing the debris and calculus, the teeth were stored in a 10% formalin solution.

The teeth were then sectioned with a slow-speed diamond disc in a transverse direction mesiodistally 1 mm below the cemento-enamel junction. The roots were removed, and the crowns were used for the investigation. Self-curing acrylic resin was poured into cylinder-shaped plastic molds. Each crown was placed in resin with the visible buccal surface facing upwards.

The buccal surface was leveled and polished gradually with 400, 800, 1,000, and 1,200 grit abrasive paper. Using adhesive tape, a 5 mm × 5 mm window of exposed enamel was produced in the center of the sample’s surface, and the samples were made resistant to acid attack by adding a uniform layer of nail polish around it. Once the samples had sufficiently dried, the adhesive tape was removed from the tooth surface using an explorer, revealing a rectangular region on the surface of the enamel.

Production of Lithothamnion Calcareum and Lithothamnion Superpositum Powder⁷^,⁸

10 gm of the respective algal extracts were used. The mineralized fronds were separated from foreign materials and debris following which they were sterilized, dried, and milled to their powdered form.

Production of Algal Solutions⁹

20 mL of 4% acetic acid was mixed with 1 gm of both the red algae separately, thereby obtaining two different algal solutions. The supernatant fluid was collected and the pH was measured. The pH was found to be 8.7.

Demineralisation Protocol⁹

Carious lesions resembling the initial stages of subsurface enamel lesions were created by incubating the tooth samples in 20 mL of demineralization bath at 37°C for 72 hours (CaCl₂ = 2.2 mm, NaH₂PO₄ = 2.2 mm, lactic acid = 0.05 m, fluoride = 0.2 ppm)

The solution was adjusted to a pH of 4.5 with the addition of 50% NaOH. This created a demineralization of approximately 135 microns in depth.

Following this the teeth were divided into their respective study groups:

Group I (n = 10) control—topical application of CPP-ACPF (GC Tooth Mousse Plus) over the demineralized teeth samples, then suspended in artificial saliva for 7 consecutive days.

Group II (n = 10)—suspended the demineralized tooth samples in a solution containing Lithothamnion calcareum for 21 hours for 7 consecutive days.

Group III (n = 10)—suspended the demineralized tooth samples in a solution containing Lithothamnion superpositum for 21 hours for 7 consecutive days.

For every 24 hours, a fresh algae solution was prepared and the teeth samples were washed twice with distilled water.

The sample specimens in groups II–III were then placed in a vibrating thermobath Rajendra Electric Motors Industries (REMI) in their respective remineralizing solutions. This simulated the oral swishing action of the saliva.

The surface microhardness was analyzed by the Vickers microhardness test. A 25 gm load was applied to each sample for a time period of 5 seconds and three indentations with a spacing of 100 microns were made. The average value of the three readings was considered.

X-ray fluorescence spectroscopy was used to determine the calcium% and phosphorus% in all samples. It is based on the principle that when individual atoms are excited by an external energy source, they emit photons of a particular wavelength. Electron beams at 2X 10^–10 amp was used and X-ray intensities in counts per second were recorded. The accelerating voltage was 15 kV.

Statistical analysis was done using analysis of variance (ANOVA) and post hoc Tukey test. The statistical analysis was done using Statistical Package for the Social Sciences version 12.0.1 for Windows.

RESULTS

The Vickers microhardness test values for all the groups are given in (Table 1). It showed that CPP-ACPF had the highest microhardness number (241.78) followed by Lithothamnion calcareum (224.35) and Lithothamnion superpositum (215.64).

**Table 1:** The mean values of the Vickers microhardness test for the three different groups
Micro hardness	N	Mean	SD	SE	ANOVA	p
CPP-ACPF	3	241.78^a	0.042	0.024	88353.08	0.001**
Lithothamnion calcareum	3	224.35^b	0.044	0.025
Lithothamnion superpositum	3	215.64^c	0.038	0.022

** Highly significant; a, b,c – Duncan Post Hoc Tukey Test

The mean calcium content for all the groups is given in (Table 2). It was found to be higher in Lithothamnion superpositum (24.88) followed by CPP-ACPF (16.82) and lastly Lithothamnion calcareum (15.77) over a period of 21 days (Figs 1 2 3).

**Table 2:** The mean values of calcium analysis by X-ray fluorescence spectroscopy for the three different groups
Calcium analysis	N	Mean	SD	SE	ANOVA	p
CPP-ACPF	3	16.82^a	0.108	0.062	18235.41	0.001**
Lithothamnion calcareum	3	15.77^b	0.076	0.044
Lithothamnion superpositum	3	24.88^c	0.076	0.044

** Highly significant; a,b,c – Duncan Post Hoc tukey test

Fig. 1: X-ray fluorescence spectroscopy for group I CPP-ACPF

Fig. 2: X-ray fluorescence spectroscopy for group II (Lithothamnion calcareum)

Fig. 3: X-ray fluorescence spectroscopy for group III (Lithothamnion superpositum)

The mean phosphorus content for all the groups is given in (Table 3). It was higher in Lithothamnion superpositum (18.42) followed by red algae Lithothamnion calcareum (14.40) and lastly CPP-ACPF (10.53) over a period of 21 days (Figs 1 2 3).

**Table 3:** The mean values of phosphate analysis by X-ray fluorescence spectroscopy for the three different groups
Phosphate analysis	N	Mean	SD	SE	ANOVA	p
CPP-ACPF	3	10.53^a	0.070	0.040	3171.65	0.001**
Lithothamnion calcareum	3	14.40^b	0.065	0.038
Lithothamnion superpositum	3	18.42^c	0.203	0.117

** Highly significant; a,b,c – Duncan Post Hoc tukey Test

Ca/P ratio for all the groups is given in (Table 4). It was higher in CPP-ACPF (1.60), followed by Lithothamnion superpositum (1.35), and lastly Lithothamnion calcareum (1.10) over a period of 21 days.

**Table 4:** The mean values of the calcium/phosphate ratio for the three different groups
Calcium/Phosphate ratio	N	Mean	SD	SE	ANOVA	p
CPP-ACPF	3	1.60^a	0.017	0.010	539.91	0.001**
Lithothamnion calcareum	3	1.10^b	0.006	0.003
Lithothamnion superpositum	3	1.35^c	0.020	0.012

** Highly significant; a,b,c – Duncan Post Hoc tukey Test

The obtained values are statistically highly significant with a p-value of 0.001 which implies it was highly significant.

The mean difference between group I (CPP-ACPF) and group II (Lithothamnion calcareum) was 17.43 and the standard deviation between the two groups was 0.002.

It was observed that all groups evaluated had tooth remineralization potential. This study also showed that though the commercial remineralizing agent had the highest remineralization potential, the alternative sources of calcium were promising.

DISCUSSION

Dental caries is a multifactorial, infectious disease caused by demineralization in the presence of fermentable dietary carbohydrates, saliva, and cariogenic oral bacteria. The dynamic caries process consists of alternating stages of tooth demineralization and remineralization, leading to the formation of discrete carious lesions at anatomical predilection spots on teeth.¹⁰

The intake of sugar gives a foundation for cariogenic microbes within the biofilm to synthesize Lactic acid. When these acids lower the pH of the biofilm to below the critical pH, the demineralization process begins. This leads to the partial or total dissolution of enamel rods.¹¹

The demineralization process is reversible as long as the acidic pH is neutralized by the buffering action of saliva The established technique for dental caries treatment is to use remineralization agents to neutralize the pH, few of the commonly used remineralizing agents include fluorides, casein calcium phosphopeptides, ozone, xylitol, and other commercially available counterparts.¹²

However, there are certain disadvantages of the commercially available remineralizing agents like they don’t penetrate deep into the enamel substructure, are expensive, fluorides cause toxicity and gastritis. CPP-ACPF has casein as one of the primary ingredients, it has been observed that there are numerous people allergic to casein.¹¹^,¹³

Over the years various natural sources of calcium have been used especially marine derivates from corals, molluscs, fishes, etc. Marine red algae have shown potential as a natural resource for calcium remineralization.¹⁴

This in vitro comparative study was done to investigate if there was any remineralization potential on tooth enamel when two red algae (Lithothamnion calcareum and Lithothamnion superpositum) were used. It also investigated whether red algae (Lithothamnion calcareum and Lithothamnion superpositum) may be superior in the quantum of tooth remineralization when compared to commercially available CPP-ACPF.

The results of this study revealed that all the groups evaluated had remineralization potential. However, CPP-ACPF had the highest remineralization potential among all the groups (241.78).

Casein phosphopeptide-amorphous calcium phosphate fluoride (CPP-ACPF) acts as a bioreservoir of calcium and phosphate ions. ACP is precipitated from the calcium phosphate solution and forms an apatite-like structure. CPP acts by stabilizing the ACP to form CPP-ACP crystals. The fluoride has a synergistic effect on the CPP-ACP to form CPP-ACPF. A study done by Reynolds et al. also revealed that the addition of 2% CPP-ACPF to the 450 ppm fluoride mouth rinses significantly increased the incorporation of fluoride into plaque.⁵ Oliveira et al. reported greater remineralization of smooth surfaces when CPP–ACP was combined with fluoride than without fluoride.¹⁵

However, among the two different red algae used, Lithothamnion calcareum was superior (224.35) when compared to Lithothamnion superpositum (215.6). Lithothamnion calcareum contains a significant amount of calcium as well as other crucial macro-minerals and microelements vital for healthy bones and joints—74 minerals including magnesium, potassium, phosphorus, iron, silicon, sodium, chloride, boron, copper, sulfur, iodine, fluoride, zinc, selenium, and strontium.⁸^,¹⁶

Raut and Gadani (2021) in their study showed that plant-based (Lithothamnion calcareum) calcium crystals were smaller isotropic crystals as compared to synthetic calcium crystals which were larger and anisotropic. Further, magnesium and boron content was higher in the case of plant-based calcium (Lithothamnion calcareum) as compared to synthetic calcium.¹⁷

Lithothamnion superpositum is rich in bone-supporting calcium, magnesium, and other minerals. The action of Lithothamnion superpositum may be attributable to the presence of other bone-supporting minerals and their effects on alkaline phosphatase, deoxyribonucleic acid synthesis, and the proliferation and mineralization of osteoblast cells.⁷

It was observed that Lithothamnion calcareum and Lithothamnion superpositum directly provided the tooth with minerals necessary for remineralization (calcium, magnesium, phosphorus, etc.) and hence could be used as an effective natural alternative for tooth remineralization.

The p-value was 0.001, between the commercial remineralizing agents (CPP-ACPF) and the alternative calcium sources; Lithothamnion calcareum and Lithothamnion superpositum.

The limitations of the study include; the cytotoxic effects of algae are unknown, there are not enough studies to know the optimum concentration and pH of the solution, and further in vitro, and in vivo tests have to be done to compare the algae to various other remineralizing agents.

CONCLUSION

In this in vitro study, the red marine algae; Lithothamnion calcareum and Lithothamnion superpositum did exhibit encouraging results in the quantum of tooth-enamel remineralization.

There are still unidentified potential compounds derived from marine algae to be discovered. Thus it can be inferred that marine algae are particularly promising for future research into bio-dental applications.

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