Effect of Nano-hydroxyapatite Crystal Incorporated Herbal Dentifrice on Remineralization of Incipient Caries Lesion- A Pilot Study

Fluoridated dentifrices are considered as an effective method for preventing dental caries. Nanohydroxyapatite crystals which have remineralizing potential can be combined with herbs with antimicrobial activity and used as a dentifrice without any risks like cytotoxicity and adverse effects. Nano-hydroxyapatite crystals are formulated by wet chemical precipitation method and tested using X-ray diffractometer. An herbal dentifrice is formulated from extract of Salvadora persica to which Nano-hydroxyapatite crystals are added. A 3x3mm wide window has been created in 14 extracted first premolars which are then processed through a pH cycling for 7 days. The depth of the lesion is assessed using a confocal microscope. The difference in mean depth of the lesion between test dentifrice (Herbal dentifrice with Hydroxyapatite) (240.33.± 23.47) and fluoride dentifrice (272.75±29.38) was found to be statistically significant. Herbal dentifrice with Hydroxyapatite group Original Research Article Kengadaran et al.; JPRI, 32(20): 13-19, 2020; Article no.JPRI.59845 14 mean depth of the lesion was 763.13+66.18 before and 240.33+23.48 after the remineralization cycle. In the Fluoride dentifrice group the mean depth of the lesion is 763.14±66.18 before and 272.55+29.38 after the pH cycle. Herbal dentifrice incorporated with hydroxyapatite had higher remineralizing potential compared to a fluoride dentifrice.


INTRODUCTION
Oral cavity is a battlefield of continuous demineralization and remineralization [1]. Enamel is the highest mineralized tissue of the body which is mainly composed of 96% inorganic material, 4% being organic material and water. However, demineralization of enamel is a common phenomenon. The ratio of demineralization and remineralization determines the hardness of the tooth structure [1,2]. The increased demineralization leads to formation of incipient lesion which initially appears as whitish spots in the enamel and progresses to dental caries [3].
The prevention and biomimetic treatment of early caries lesions, particularly in individuals at high risk for developing caries, which is a prevalent chronic oral disease worldwide, are on-going challenges for the dental research and public health communities [4]. Also the prevention of carious lesions is cost effective compared to restoration. Proper brushing may aid in maintenance of good oral hygiene thereby reducing the incidence of carious lesions [5].
Several studies have proven that dental caries in its early stage of formation can be remineralized, and this remineralization can be facilitated by such agents as fluoride, delivered via either mouth rinse or dentifrice [6]. However, extensive use of fluoride mainly in the form of a dentifrice has contributed to a rising incidence of dental fluorosis [7]. Hence, there is a need for an alternative caries remineralizing and preventive agent as effective as fluoride. In recent times, there has been renewed interest in naturally occurring products [8,9]. Several studies have shown that miswak (Salvadora persica) has significant antimicrobial activity against both aerobic and anaerobic bacteria [10,11]. However, the remineralizing capacity of the herb is questionable. Hydroxyapatite is an important source of calcium and phosphate, very important for the remineralization of demineralized enamel areas [12,13].
Incorporation of hydroxyapatite crystals with Salvadora persica may have a synergistic effect against dental caries with both antimicrobial efficacy and remineralization capacity. According to the epidemiological triad for the dental caries causes, there is a requirement to evaluate the host factors such as oral health knowledge, oral hygiene practices, dental visits, and eating habits among the school going children.We have successfully completed numerous epidemiological studies [14][15][16][17][18][19][20]21] and in vitro studies [22,23,24,25] for the betterment of our community [26,27,28,29,30,31] Hence a study was planned to compare the remineralizing effect of hydroxyapatite incorporated herbal dentifrice and conventional fluoridated dentifrice on artificially created enamel lesions.

MATERIALS AND METHODS
An in-vitro experimental study was conducted in the Department of Public Health Dentistry. The sample size was estimated to be 7 per group based on the study done by Roza Haghgoo et al., using G power software version 3.1 with a power of 95%. Ethical committee approval was obtained prior to the start of the study. Extracted sound human maxillary premolar with enamel defects, dental fluorosis, fracture or micro cracks (under magnification of 4X), wasting diseases and caries or restorations were excluded from the study.

Pre-study Procedures
Teeth were collected and stored in a 10% formalin solution until further use. The buccal surface of each tooth was coated with acid resistant nail varnish leaving a window of 3×3 mm. This was done to limit the area of demineralization and remineralization only in the window area.

Preparation of Solution
The buffered de-/re-mineralizing solutions were prepared using analytical grade chemicals and deionized water.

Preparation of Toothpaste
Nano-hydroxyapatite crystals are formulated by wet chemical precipitation method and tes using X-ray diffractometer (Fig.1).

pH Cycling
The pH-cycling models are used to stimula dynamics of caries formation by inducing demineralization and remineralization cycles (Fig. 3).

Depth of the Lesion Assessed after pH Cycling
The treated enamel blocks to be then analyzed using confocal microscope. These values are noted, tabulated and compared with sound and demineralized zones of the same specimen. Thi is to be done for two groups (Fig. 4)

Statistical Analysis
Normality of the data was assessed using Shapiro Wilks and Kolmogorov Smirnov test. Data to be entered in Microsoft excel spreadsheet and analyzed using SPSS sof (version 21). Independent 't' test was used to compare between the groups. Mean imputation was done to replace the lesion depth on demineralization.

Depth of the Lesion Assessed after
The treated enamel blocks to be then analyzed using confocal microscope. These values are tabulated and compared with sound and demineralized zones of the same specimen. This 4).
Normality of the data was assessed using Shapiro Wilks and Kolmogorov Smirnov test. Microsoft excel spreadsheet and analyzed using SPSS software 't' test was used to compare between the groups. Mean imputation was done to replace the lesion depth on

RESULTS AND DISCUSSION
Dental caries is one of the most prevalent chronic diseases of people worldwide; individuals are susceptible to this disease throughout their lifetime [13,32]. Affected teeth cannot be saved always and may have to be extracted when extensively damaged. Brushing is an important in-house method of maintaining oral hygiene which helps in preventing dental caries. Often this mechanical plaque control method of brushing is associated with chemical plaque control of using toothpaste [33].
Fluoride is an important ingredient in dentifrices. Nearly 80 years on and fluoride remains one of

RESULTS AND DISCUSSION
Dental caries is one of the most prevalent diseases of people worldwide; individuals are susceptible to this disease throughout their . Affected teeth cannot be saved always and may have to be extracted when ed. Brushing is an important house method of maintaining oral hygiene which helps in preventing dental caries. Often this mechanical plaque control method of brushing is associated with chemical plaque Fluoride is an important ingredient in dentifrices. Nearly 80 years on and fluoride remains one of dentistry's key strategies for the prevention of dental caries [33,34]. However, Fluoride containing toothpaste can be toxic if swallowed in large amounts [35]. Hydroxyapatite is an important source of calcium and phosphate, which helps in remineralization of demineralized enamel areas [33,34]. Salvadora strong antimicrobial effects against cariogenic microorganisms [36]. Formulating a dentifrice with nano-hydroxyapatite crystals (n Salvadora persica as active ingredients may have a synergistic effect. Herbal dentifrice with Hydroxyapatite showed a statistically significant difference in lesion depth before and after demineralization and remineralization cycle. The mean depth of the lesion was 763.13 the confocal microscopic image of demineralization and remineralization dentistry's key strategies for the prevention of . However, Fluoridecontaining toothpaste can be toxic if swallowed in Hydroxyapatite is an nd phosphate, which helps in remineralization of demineralized Salvadora persica has strong antimicrobial effects against cariogenic . Formulating a dentifrice hydroxyapatite crystals (n-HA) and as active ingredients may have a synergistic effect. Herbal dentifrice with Hydroxyapatite showed a statistically significant in lesion depth before and after demineralization and remineralization cycle. The mean depth of the lesion was 763.13+66.18

Comparison of depth of the lesion between two different groups
Comparison of depth of the lesion between two different groups p-value 0.043 *  (Table 2). In the Fluoride dentifrice group the mean depth of the lesion is 763.14±66.18 before and 272.55+29.38 after the pH cycle. The difference was found to be significant statistically (Table 3). Thus, herbal dentifrice with Hydroxyapatite (240.33.± 23.47) had better remineralizing effect than fluoride dentifrice (272.75±29.38). Both the dentifrices exhibited remineralizing effects following the pH cycle. However, the results of this study could not be compared due to lack of similar studies.
Limitations of this study may be that the tooth may have greater susceptibility than others due to the age of donors and exposure to environmental factors like fluoride and the amount of demineralization in the cycle is much greater than in the oral environment. Hence, no in-vitro study can replace an in vivo study.
Further in vivo-studies have to be done to evaluate the combined anticariogenic and antimicrobial effectiveness of the Salvadorapersica and nano-hydroxyapatite crystals.

CONCLUSION
A herbal dentifrice with Salvadora persica and nano hydroxyapatite crystals as an active ingredient was formulated and remineralizing effectiveness was assessed using confocal microscopy. Both fluoride dentifrice and herbal dentifrice with hydroxyl apatite have remineralizing effectiveness. However, the newly formulated herbal dentifrice with nano hydroxyapatite crystal was found to be more effective in remineralizing the artificial caries lesion.

CONSENT
It is not applicable.