Evaluation of Remineralisation Potential of Zingiber of icinale Roscoe-Apis Mellifera, and Chitosan as compared to control using QLF on white spot lesions: An in-vitro study

Early detection and treatment of white spot lesions (WSL) is pivotal in caries control. Several commercially available products are available for WSL reversal. However, themajority of them are either synthetically derived or are not a hundred percent ef icient. Thus there is an ever constant need to ind newer, more ef icacious products for the same. One of the parameters to quantify de and remineralization is by Quantitative Light Induced Fluorescence (QLF). Thus this study aims to evaluate and compare the remineralizing potential of Zingiber of icinale Roscoe (Ginger rhizome), Apis Mellifera (Manuka Honey) mixture and chitosan on arti icial demineralized human enamel using Quantitative Light Induced Fluorescence. 45 human enamel samples were randomly divided into a control and two test groups. An Area of Interest (AOI) measuring 4x4 mm on the buccal surface of each tooth was formed and all the samples were subjected to demineralization process for a period of 96 hours. Remineralization regime was then carried out with twice daily application of respective interventional agents for a period of 21 days. QLF readings were recorded at the end of demineralization (Baseline), Day 7, Day 14 and Day 21 and luorescence imageswere analysed using QLF InpektorTMpropriety software. The remineralization action of chitosanwas found to be the highestwith a statistically signi icant reduction (p<0.001) seen at the end of 7th, 14th and 21st day. Gingerhoney mixture also showed reduction in luorescence levels but only after the 14th day. The current study showcases the dramatic ability of chitosan to almost completely reverse arti icially formed WSL at each subsequent week. Ginger honey on the other hand showed a signi icant remineralization between2nd and3rdweekwhichmaybe attributed to a possible slower mechanism of action.


INTRODUCTION
The white spot lesion (WSL) is the irst clinically apparent sign of an otherwise silent disease-that is dental caries. Gocmen de ined the WSL as "a subsurface enamel porosity from carious demineralisation" that is manifested clinically by a milky white opacity (Gocmen et al., 2016). Caries detection clinically is still largely dependent on visual and radiographic examination. However, these methods can only detect well-advanced lesions, involving at least 300-500µm of enamel. Thus, WSL or non-cavitated lesions, where a non-surgical reversal is still possible, are dif icult to identify using these methods alone. Quantitative Light-Induced Fluorescence or QLF was introduced clinically as a caries detection system in 2004 (QLF-Pro Inspektor, Germany). It works on the principle that enamel will auto luoresce under certain light conditions. Demineralised enamel will luoresce less and this loss of luorescence can be detected, quanti ied and longitudinally monitored using QLF. Studies have shown QLF to have high sensitivity for quanti ication and monitoring of de and remineralisation with high correlation to changes in mineral content (Shi et al., 2001;Pretty et al., 2002).
Current paradigms in minimal intervention dentistry especially for WSL treatment show a trend of non-surgical treatment with a greater importance being given to prevention and reversal of disease process over cure; remineralisation of WSL being one such natural repair process.
Several synthetic agents are available to mineralise early enamel carious lesions. However, naturally derived products are known to show lesser toxicity and are considered 'Generally Recognised As Safe' (GRAS) by the US Food and Drug Administration (FDA) (Summitt et al., 2006).
Chitosan, a derivative of chitin, is well known for its use in wound dressings and drug delivery systems (Agnihotri et al., 2004;Kumar et al., 2004).
Its increased drug targeting potential is ascribed to improved drug absorption and stabilisation of the drug components.
However, the remineralising potential of deacetylated Chitosan alone in the presence of arti icial saliva has not been assessed. Ginger and Manuka Honey are naturally derived products that show exceptional antibacterial activity, especially against oral bio ilm (Park et al., 2008;Patel et al., 2011;Azizi et al., 2015). Ginger rhizome also has a high luoride content making its use in remineralisation therapy hypothetical. Thus, the present study aimed to assess the remineralising potential of 90% deacetylated chitosan and ginger-manuka honey mixture in the presence of arti icial saliva on arti icially formed incipient enamel lesions.

Ethical Clearance & Study design
The procedure protocol was approved by the Institutional Research Ethics Committee (JSS/DCH/IEC/MD-26/2016-17(2)) before the commencement of the study. This experimental in-vitro study was conducted in the research unit of our institution. The demineralising solutions and test solutions were prepared in the College of Pharmacy.

Preparation of Demineralising Solution and artiicial saliva
2 Litres of the demineralising solution was freshly prepared every day according to the composition given by Featherstone and Zero (1992). 4 Litres of arti icial saliva was made once in every two days, according to the compositional structure given by Sato et al. (2006).

Collection and Identi ication of Ginger
The ginger rhizomes were collected from the northern part of Haryana in Jhajjhar district. The ginger rhizomes were identi ied and classi ied in the Department of Biological sciences.
The rhizomes were washed with clean water and allowed to air dry to reduce the microbial load of the plant material due to handling and transportation.
The outer covering of ginger was peeled, and the rhizomes were allowed to sun dry for two weeks. The dried ginger rhizomes were cut and pulverised into powder using an electronic blender.

Collection of Honey
The honey was harvested in Taranaki, in the westcentral part of the North Island of New Zealand in the summer-early spring of 2016 and consisted mainly of nectar gathered from the blossoms of Manuka trees.
The extracted honey from combs neither contain any preservatives nor went through any preservative processing. Extraction, storage and transportation of Manuka honey was done in glass containers.

Preparation of ginger honey mixture
The Ginger powder was mixed with Manuka honey (MGO activity of 580) in a ratio of 8mg/ml (w/v) (Bilgin et al., 2016).
All the interventional agents were freshly prepared daily.

Collection, storage & Preparation of human permanent enamel samples
For the present study, enamel samples were obtained from 45 human premolars freshly extracted for orthodontic reasons; free from dental caries, fracture, hypoplastic lesions, intrinsic stains, wasting diseases like attrition, abrasion, erosion, developmental anomalies and restorations.
Immediately after extraction, the teeth were stored in 10% formalin and were thoroughly cleaned of debris, calculus and soft tissues.
They were washed in 0.1M Phosphate buffer (pH 7.4), rinsed with de-ionised water and were stored in distilled water at a temperature of 4º C until further use (Shanbhog et al., 2016).
The premolars were embedded in clear acrylic blocks measuring 2 x 2.5 x 1 cm using a customised jig. The middle third of the buccal surface of each tooth was covered in a polyvinyl stencil measuring 4 x 4 mm to form an Area of Interest (AOI).
Each sample was then painted with two coatings of colourless acid-resistant nail varnish (Colour Plus, MaybellineTM) barring the 4 x 4 mm window.
AOI was considered for the analysis of mineralisation and lesion progression (Mehta et al., 2013).

Deminereralisation of enamel samples
Each sample was placed in sterile containers containing 30ml of the demineralising solution and placed in an orbital shaking incubator at 37 º C at 50 RPM for 96 hours. Immediately after the demineralisation cycle, baseline QLF readings were done. Only teeth with an ICDAS score of 1 or 2 after demineralisation were included in the study.
The demineralised samples were then randomly assigned to either Control, Ginger-Honey or Chitosan groups and subjected for remineralisation cycle.

Reminereralisation Cycle
The demineralised samples were subjected for 21 days of remineralisation cycle. All the samples from three groups were kept immersed in 30ml of arti icial saliva for the entire duration of the study. The control group was treated with distilled water, and the two experimental groups were treated with ginger honey mixture and Chitosan, respectively. All samples were subjected to the 1-minute application of respective interventional agents twice a day for 21days.

QLF analysis
QLF analysis was carried out by a single examiner using QLF-D Biluminator T M device (Inspektor). QLF and white light digital images were captured from buccal aspects of the specimens under class 1 ASA darkroom conditions (3) at baseline, 7th day, 14th day and 21st day of remineralisation. Lesion depth (∆F), Maximum luorescence loss (∆F Max) and lesion volume (∆Q) were recorded and analysed using QA2 v 1.26, Inspektor Research Systems software.

Data Presentation and statistical analysis
The collected data were coded in EXCEL and analysed using SPSS Version 23. For data presentation, mean values and standard deviations of ∆F, ∆F Max, ∆Q were calculated. Data analysis was performed using Wilcoxon signed ranks test and repeated measures of ANOVA followed by Tukey's post hoc test.

RESULTS
Mean difference values of lesion depth (∆F), Maximum luorescence loss (F Max) and Lesion volume (∆Q) for Control, Ginger-Honey and Chitosan groups at different time intervals of remineralisation cycles were obtained. Table 1 depicting mean lesion depth (∆F), showed a reduction in all the three groups across different time intervals. However, the chitosan group showed a statistically signi icant luorescence gain at 7th, 14thand 21stday of remineralisation cycle as compared to baseline (p-0.001). Ginger-Honey showed statistically signi icant luorescence gain at 21stday of remineralisation cycle as compared to 14th day (p-0.001).
Mean Maximum luorescence (F Max) loss also reduced in the three groups across different time intervals. Control, Ginger-Honey and Chitosan Groups showed statistically signi icant luorescence gain at 21st day of remineralisation cycle as compared to baseline (p-0.001, 0.004 and 0.003 respectively). Mean Lesion volume (∆Q) reduced in the three groups across different time intervals. Control and Chitosan Groups showed statistically signi icant luorescence gain at 21stday of remineralisation cycle as compared to baseline (p-0.031 and respectively).
Comparison of remineralisation potential of Ginger-Honey with Control across the period showed a statistically non-signi icant difference. Table 2 Comparison of remineralisation potential of Chitosan with Control across the period showed a statistically signi icant difference between baseline to 14thand 21stday (p-0.041 and 0.001). Table 3 Comparison of remineralisation potential of Chitosan with Ginger-Honey across the period showed a statistically signi icant difference between baselines to 21st day (p-0.003). Table 4 DISCUSSION Reminereralisation treatment protocols are based on the physiological phenomenon of mineral loss and gain and changing the balance between the two. Various preventive therapies have been studied to enhance remineralisation, reduce demineralisation and to arrest active carious lesions-luoride being the most commonly used amongst them (Pulido et al., 2008). Fluoride levels of about three parts per million (ppm) in the enamel are required to shift the balance from net demineralisation to net remineralisation (Pretty et al., 2002). However, normal remineralisation by luoride is found to be a self-limiting surface phenomenon that prevents the penetration of ions into the depth of the lesion (Cate, 1990). Rapid deposition of a surface layer of luorapatite is not only observed to resist demineralisation but also prevents any further penetration of calcium and phosphate ions. Thus 100% reversal of the incipient lesions is not possible with luoride alone. Several materials like Stannous Fluoride, Casein Phosphopeptide, Casein Phosphopeptide-Amorphous Calcium Phosphate, Casein Phosphopeptide-Amorphous Calcium Phosphate Fluoride etc. were brought into the market with varied results (Reynolds et al., 1995;Cochrane et al., 2008) to overcome these shortcomings. Natural products have been used with dental formulations like mouthwashes, irrigating agents, intra-canal medicaments, anti-in lammatory etc. However, only an exiguous number of the commercially available remineralising agents are naturally derived. The current study used 90% deacetylated Chitosan and an experimental formulation of ginger and Manuka honey to test their remineralisation potential in vitro against control. Chitosan and its derivatives have well documented biological activity and are used in medicine mainly as a drug delivery system. Majority of the remineralisation studies have used Chitosan in combination with various interventional agents. Some of the interventional agents are phosphorylated Chitosan, chitosan pre-treatment with bioglass and chitosanamelogenin hydrogel (Ruan et al., 2013;Zhang et al., 2018;Xu et al., 2011). Various mechanisms of action have been proposed for the same (Hayashi et al., 2007;Decker et al., 2005). Chitosan has been found to have the ability to bind calcium ions to form nucleating sites, and it also gets adsorbed to the surface of hydroxyapatite crystals which helps in the formation of nano-complexes thereby leading to remineralisation (Xu et al., 2011;Lee et al., 2012). The present study made use of Chitosan alone and tested its ability to successfully deliver calcium and phosphate ions from arti icial saliva to the inner enamel layers. Study results showed statistically signi icant luorescence gain across week 1, 2 and 3 with 90% deacetylated chitosan intervention in the presence of arti icial saliva (p<0.001) leading to substantial remineralisation of lesion across the study period. Manoj (2007) and Nigus and Chandravanshi (2016) have shown dried ginger rhizome to have a luoride concentration ranging from 2.0-2.8 mg/kg. Gingerhoney group in the present study showed minimal luorescence gain from baseline to week one and from week 1 to week 2. However, between week two and week 3, a highly signi icant luorescence gain was noticed (p<0.002). This may be attributed to a possible slower mechanism of action. Further studies with a more extended study period and remineralisation cycle are advocated for the same.
This result was found con licting with that of previous studies wherein a signi icant amount of remineralisation was noticed on the usage of gingerhoney mixture (Gocmen et al., 2016;Korkut et al., 2017). This could be attributed to mineral variations in the soil, irrigation water, and the atmosphere, and differences in the agrochemicals used during cultivation, such as fertilisers, pesticides, and herbicides.

CONCLUSION
It can be concluded taking into consideration the limitation of the present in vitro study, that: Compared to ginger honey mixture, Chitosan produced signi icant amounts of remineralisation of the artiicial white spot lesions. Chitosan produced significant amounts of remineralisation at 7th, 14th and 21st day as compared to only 21st day for arti icial saliva and ginger honey groups. Ginger honey showed signi icant remineralisation between 2nd and 3rd week , possibly due to a slower mechanism of action. Study with an increased remineralisation period is advocated for identi ication of the same.