The in vitro quantitative observational research study was carried out in the Department of Conservative Dentistry and Endodontics, Vinayaka Missions Sankarachariyar Dental College, Salem, Tamil Nadu state, India for one month duration from June 20, 2020 to July 20, 2020. The ethical clearance number was obtained (VMSDC/IEC/Approval No.160).

Synthesis of Calcium Carbonate Powder

The Calcination procedure was performed to collect pure powder free of impurities and to elevate the alkalinity mussel shell powder. Commonly mussel shell extract powder possesses 95% calcium carbonate, which transforms to basic calcium oxide on calcination, and this is responsible for the increase in alkalinity [8]. The mussel shells were obtained from Malpe beach, Mangalore, India. The calcium carbonate powder was prepared from the mussel shells according to the method explained by Islam KN et al., [9]. Specimen of the mussel shells were dehydrated in an oven at 50°C for 7 days, and then crushed and amalgamted into a refined powder, which was filtered through a 90-μm laboratory stainless steel sieve (Jai Instrument and Co, Chennai, India). The powder was measured for its discrete elemental composition percentage by weight using X-ray fluorescence spectroscopy analysis (Spectrace 6000 USA).

Synthesis of Mussel shell Powder Extract Solution

One gram of MSPE dissolved in 20 mL of 4% acetic acid (India Chemicals, Pvt., ltd., Mumbai, India) in a test tube. The clear fluid which is formed at the top was moved to a beaker and the pH of the solution was measured using a pH meter (Deluxe deep vision, model no: 101, California, USA) which was 11.8.

Preparation of sample: Sampling technique applied was purposive sampling technique, power of study not applicable in the study. Five recently extracted unerupted lower third molars were cleaned and decoronated at CEJ, followed by sectioning single tooth longitudinally in a mesio-distal and bucco-lingual direction with diamond saw such that four samples collected from a single tooth. Enamel blocks of four different dimensions (4 mm length, 4 mm width and 2 mm thickness) were taken out of each tooth and then were inserted into the acrylic blocks.

Demineralisation Protocol

Early phase of caries in the form of subsurface enamel lesion was obtained by inserting sectioned tooth samples in 20 mL of artificial carious solution (Demineralisation solution) for 72 hours (CaCl₂=2.2 Mm NaH₂PO₄=2.2 Mm Lactic acid=0.05 M, Fluoride=0.2 ppm, solution was regulated with 50% NaOH to a pH of 4.5) [10,11]. The sectioned tooth samples inserted in the demineralisation solution (CaCl₂, NaH₂PO₄, Lactic acid and Fluoride) for 72 hours at 37°C formed a subsurface demineralisation of almost 150 microns width with an intact surface exhibiting an early enamel lesion [11,12]. The observations were conducted using SEM (Hitachi SU-1510; Hitachi High-Technologies Corp., Tokyo, Japan) under 20 kV and 50× magnification.

Study contained homogeneous samples and sample size was calculated using Purposive sampling technique. The complete sample of 20 enamel blocks divided into two groups:

Group A (n=10) Experimental Group- subsurface demineralisation followed by inserting the tooth samples in mussel shell extract solution for 24 hours for 30 successive days for remineralisation. For every 24 hours, fresh mussel shell extract solution was prepared and the samples purified twice with distilled water.

Group B (n=10) Control Group- Topical application of Clinpro (3M ESPE, Saint Paul, MN, USA) was performed after subsurface demineralisation and then placing the tooth samples in artificial saliva for 24 hours for 30 successive days for remineralisation. For every 24 hours, fresh artificial saliva was prepared and topical application of clinpro done.

Microhardness Testing

Samples were collected by single trained examiner. Training and calibration was done before the start of the study and it was found to be 0.83 which indicates excellent agreement for single examiner on two different occasions. Unit for Microhardness test is micrometre (μm). Vickers microhardness testing machine (Lieca, chu-linh, Japan) was used to test the surface microhardness. A load of 25 g applied for 5 seconds and five indentations was applied for single sample with a spacing of 100 microns [11]. Microhardness testing was done in Alpha omega Hitech Bio Research centre, Salem, India.

Atomic Analysis by Energy Dispersive X-ray Spectrometry

Energy dispersive X-ray spectrometry (Quanta 200 FEG) was employed in all samples to analyse both calcium and phosphorus ions. Electronic beams sustained at 2 x10-10 amp and X-ray magnitude in counts per second was calculated.

Data were computerised and analysed using Statistical Package for the Social Sciences (SPSS) version 22.0. Unpaired t-test was used to compare mean scores of both groups.

Statistical analysis using unpaired t-test was used to check differences for Microhardness (Vickers microhardness testing machine) and atomic analyses (Energy Dispersive X-ray spectrometry) between groups at the level of p≥0.05 which was considered statistically significant.

Clinpro exhibited statistically higher microhardness value than MSPE as shown in [Table/Fig-1].

[Table/Fig-2] depicts no statistical difference in calcium/phosphate ratio value for both groups.

X- Ray Fluorescence Spectroscopy Analysis

Chemical analysis of Mussel shell powder extract using X-ray fluorescence spectroscopy demonstrates highest calcium concentration of 95% and 0.46% of phosphate. It also shows 0.53% of Magnesium, 0.18% of Strontium, 0.11% of Sulfur and 0.03% of Potassium. The results exhibited no statistical difference in quantitative amounts of Ca weight % and P weight % for both groups as shown in [Table/Fig-2].

Remineralisation process of enamel is reinforced by the amount of calcium content that exist on tooth surface. MSPE has a good percentage of bio-available calcium. In late years, mussel shell powder extract has accomplished importance in various fields. The chemical analysis of MSPE performed with X-ray fluorescence spectroscopy assessed that it comprises 95% calcium. Lately, many studies have stated the uses of mussel shell like calcium oral supplements [13-16]. Calcination was carried out in this study to increase the alkaline nature of the powder and make it free from pathogens [11]. Additionly, 10% acetic acid was added so that the mussel shell powder is nearly devoid of pathogens [8]. This was an in-vitro research study and the first of its type to assess the remineralisation potential of early enamel carious lesions by MSPE solution. MSPE solution was prepared by dissolving one gram of MSPE in 20 mL of 4% acetic acid (India Chemicals, Pvt., ltd., Mumbai, India) in a test tube. The clear fluid which is formed at the top was moved to a beaker and demineralised tooth samples were suspended in it. The MSPE solution can also be applied to the tooth surface with help of different vehicles such as glycerin solution or methylcellulose gel in an application.

In a study done by Lata S et al., demineralisation solution produced a subsurface deminerlisation of width of 150 microns and the surface was intact like an early enamel lesion. The calcium and phosphate (50% saturation) present in the solution caused dissolution of enamel subsurface only [12].

Surface demineralisation was blocked by deposition of fluoride and forming fluorapatite at the surface [12]. Superior remineralising ability on surface and subsurface lesions of enamel was exhibited by Clinpro Tooth Creme involving Tri Calcium Phospate (TCP) with 950 ppm fluoride, as proved by the manufacturer. Calcium fluoride co-exist in a protective barrier at the time of manufacturing process. This will help in movement of TCP to the teeth. When it combines with saliva during brushing, the barrier breaks and lead to calcium, phosphate and fluoride easily accessible to the tooth. This arrests the demineralisation and promotes remineralisation [8].

In this study, Clinpro displayed statistically better remineralisation property by exhibiting more hardness value than MSPE. When linked with another natural calcium sources, MSPE has low levels of toxic metals like Pb, Al, Cd, and Hg. The N-terminal sequence of mussel shell matrix proteins will promote in increased calcium transport and regarded as a potential significance of mussel shell when consumed as calcium supplements [16]. Hence, MSPE solution was used in this study.

The pH of a MSPE solution was tested by pH meter which was 11.8. The raised pH of a remineralising solution is indicated, as it upgrades the ion activity of anions such as phosphate and hydroxyl ions in the solution. The ionic activity relates to the concentrations of these ions in the solution. Asian green mussel (Perna viridis) shells most commonly found and they are most often discarded after consumption and can therefore be acquired with minimal economic incentives. Since, there’s increased availability of these shells, it will be easy to evaluate enamel remineralisation potential effectively.

Since, this study is an in vitro quantitative observational research study, Experimental group samples were immersed in MSPE solution to evaluate effectiveness to remineralisation and control group samples were immersed in artificial saliva solution.

At low pH, there will be increased H+ ions, which will bind with these anions making them less accessible for remineralisation. In addition, the basic form of phosphate anion present in hydroxyapatite is PO₄^-3 and these anions are present in higher concentrations only at a high pH of 11-12. For remineralisation to occur, bioavailable calcium and phosphates are beneficial [17]. Therefore, the increased bioavailability of calcium along with the raised concentration of phosphates present in MSPE solution in conjunction with its higher pH may be responsible for remineralisation.

The lacunae in the area of study are some difficulties in sterility and removing all contaminants from the surface of marine skeletal species.

Within the limitations of this study, it can be concluded that the raised pH of the of mussel shell powder extract solution in addition to rich bioavailable calcium content of mussel shell has the efficiency to favour remineralisation. Even though Clinpro showed more remineralisation than MSPE, the latter due to its easy bioavailability and natural source of calcium and phosphate can be the future in remineralising enamel carious lesions. Further, clinical studies regarding suitable vehicle for MSPE is required as this might raise the remineralisation potential of MSPE comparable to the commercially available agents.

Financial aid: Authors are extremely thankful for supporting this work by Vinayaka Mission’s Research Foundation - (Deemed to be University) Unique ID: VMRF/Seed Money/2020/VMSDC-Salem/10.

Authors contribution: VDR and RS designed project and computational framework, VDR performed the experiments and derived the models, YCT and EMI assisted with collected measurements and helped to carry out the collected simulations, VDR wrote the manuscript in consultation with other authors.