AN IN VITRO STUDY ON THE EFFICACY OF FOUR REMINERALIZING AGENTS

Saravanakumar Subramanian¹, Prema Anbarasu^1*, Anitha Nallusamy¹, Bharat Ramesh¹

¹Department of Orthodontics, Chettinad Dental College and Hospital, Chennai, India. [email protected]

ABSTRACT

To equate and assess the four commercially potential remineralization available products namely GC Tooth Mousse, SHY-NM, Biomed calcimax, and Bentodent on demineralized human teeth. 72 upper premolars bonded with 3M premolar bracket were included in this study. Teeth were randomly selected and divided into six groups of 12 teeth each. Group 1: Stored in de-ionized water alone (Positive control); Group 2: Stored in Tencates demineralizing solution alone (Negative control); Group 3: Stored in Tencates demineralizing solution & coated with GC Tooth Mousse every 4 hours; Group 4: coated with SHY-NM; Group 5: coated with Bio med calcimax; Group 6: coated with Bentodent. After 96 hours teeth were sectioned using a diamond disc and then thinned to 150-200 um thick with carborundum stone. Every segment acquired was envisaged underneath a polarized light microscope and analyzed using Image J software. Bonferroni Post-hoc test for multiple pairwise comparisons for minimum demineralization depth reveals a significant difference in the depth of demineralization except, for Group 3 and group 4; Group 5 and group 4. Post-hoc test for maximum demineralization depth reveals a significant difference between, Group 2 and group 6; Group 3 and group 4; Group 4 and group 5; Group 5 and group 6. GC Tooth Mousse & SHY-NM are effective in remineralizing artificially induced caries lesions compared to Biomed calcimax and Bentodent.

Key words: Enamel demineralization, White spot lesion, Medicaments, Polarized light microscop.

Fixed orthodontic appliances increase the prevalence of white spot lesions (WSLs) regardless of prophylactic measures & have a negative impact on the aesthetic appearance of the tooth [7]. The novel approach in caries management is the non-invasive method (i.e.) enamel remineralization which is one of the most exciting topics for researchers for the past 100 years. This method can change the lesion from an active to an inactive state. Toothpaste, varnishes, gels, and fluoride-releasing materials are commonly used professional delivery methods, to remineralize high-risk areas. The primary mechanism of remineralization occurs by diffusion of ions especially calcium & phosphate from saliva and topical agents to reconstruct an acid-resistant, hypermineralised layer on the existing crystal remnants which then act as remineralization nuclei [8]. There are many techniques to measure mineral changes in human enamel. These techniques include microhardness measurement of enamel cross-sections, different microradiography techniques, polarized light microscopy, iodine absorptiometry, and light scattering. In this study, Polarized light microscopy (PLM) analysis was selected as it is extremely sensitive to changes in hard tissues [9]. Various remineralizing agent has been presented in current years as additives for traditional fluoride-based systems [10]. Hence, this in vitro survey was assumed to assess and equate the remineralizing potential of four commercially available products namely GC Tooth Mousse, SHY-NM, Biomed calcimax, and Bentodent on demineralized human teeth. GC Tooth Mousse contains CPP-ACP, SHYNM contains bioactive glass, Biomed calcimax contains calcium hydroxyapatite and L-Arginine, and Bentodent toothpaste contains calcium bentonite. In this study, 72 Upper human premolar teeth indicated for therapeutic extraction were collected and stored in 10% formalin because it acts as the best disinfecting agent & also it resists demineralization by fixing proteins in the organic pellicle attached to the surface of teeth during storage [11]. In experimental caries studies , Spontaneously formed WSLs to be used. Nevertheless, it is tough to produce a carious lesion in vivo in standard sizes. Consequently, a demineralization solution was selected in this study to form incipient caries lesions of standard size. The teeth were kept in this solution for 96 hours as described by Reynolds and Black [12].

To simulate the dynamic process of demineralization & remineralisation, Ph cycling model was adopted in many studies.  However, Ph Cycling models have limitations like the inability to simulate the intraoral conditions progressing to caries development; to mimic plaque fluid & salivary composition experienced in vivo and to simulate topical use & clearance of product from the oral cavity[13].

Saumya Kakkar et al. used Tencates demineralization solution alone in their study [14]. Although the same limitations apply to the usage of chemical demineralization alone, it was the preferred method of choice due to its simplicity and practicality. Group 1 was immersed in de-ionized water due to previous studies published that de-ionized water does not alter the microhardness of the teeth significantly till 2 months [15], whereas the remaining five groups were immersed in Tencates demineralizing solution for 96 hrs at room temperature [16]. The lesion depth was evaluated using a Polarized light microscope due to the histological characteristics of enamel and dentin can be visualized better than transmitted light microscope due to its birefringence property,

In the present study, group 3(10% CPP-ACP -Recaldent- a water-based, lactose-free cream) showed increased remineralization when compared to group 4, group 5 & group 6. Recaldent technology was developed by Eric Reynolds and co-workers at the University of Melbourne, Australia. Previous studies have shown a short term remineralising effect and long-term caries-preventing effect in the in vivo clinical and randomized control trial [17].

Two studies concluded that CPP-ACP showed better remineralising potential than calcium sodium phosphosilicate using scanning electron microscope (SEM) and energy dispersive x-ray analysis (EDAX) [18, 19]. Four weeks of application of CPP-ACP, after debonding resulted in a significant reduction of white spot lesions. Hence CPP-ACP is effective before, during or after acid intervention [20, 21]. Moreover casein phospho peptide-amorphous calcium phosphate decreased mutans streptococci and showed a reduction in the bacterial count [22].

Following CPP-ACP, the effective material in the study was SHY-NM which contains bioactive glass (Novamin). A study using the Vickers micro hardness test concluded that initially Novamin showed superior remineralizing properties than CPP-ACP but eventually both have similar remineralizing potential [23]. On bleached enamel BAG act as a reservoir of ions in demineralized areas. A similar study concluded that novamin resulted in the formation of a protective layer on the enamel surface after 10 days of the remineralization phase [24]. A study using a scanning electron microscope revealed that BAG plugs are larger, angular, and intimately attached to the enamel surface compared to the plug formed by CPP-ACP [25].

The next remineralizing agent following CPP-ACP, SHY-NM was biomed calcimax toothpaste. Without additional additives, synthetic HAP particles are attached to pellicle-covered enamel surfaces and adhesion is directly proportional to the organic enamel content and inversely proportional to its size (1.3um). By filling small pores in demineralized tooth surfaces, the HAP (Hydroxy apatite) crystal stimulates remineralization [26].

The dipole properties of HAP and the resulting electrostatic forces are responsible for the cohesion of tooth surface crystallite clusters that provide the effect of antibiofilm and minimize solubility. HAP replaces fluoride and antimicrobials in a dentifrice & it is more effective for xerostomic patients [27]. HAP crystals improve enamel remineralization and resistance against acids even after three days and three months. A study using Vickers microhardness tester & SEM concluded that remineralizing efficacy of n-HAP toothpaste was higher than that of BAGs toothpaste due to the smaller particle size of nanohydroxyapatite (50-1000 nm) compared to the hydroxyapatite (1.3 um) [28].

Calcium bentonite is considered to have least remineralizing potential. A phyllosilicate group of minerals named after Montmorillon in France is bentonite or montmorillonite. Montmorillonite (MMT) is layered clay formed by silicon oxide (tetrahedral structure) and aluminum hydroxide (2:1) (octahedral structure).

These particles are plate-shaped with an average diameter of around 1 um. The replacement of lower valence cations, which leaves the adjacent oxygen atoms with a net negative charge that can attract cations, is known as cation exchange capacity (CEC). There is continuous attraction of calcium & phosphate ions via Cation Exchange Capacity (CEC), resulting in supersaturation & remineralization [29].

Recently Calcium montmorillonite is used as fillers in dental composites to improve remineralizing potential [30]. There are no studies to provide data about the remineralizing potential of calcium bentonite as toothpaste.

Limitations of this study includes inability to simulate the oral environment and the Protective potential .Only quantitative changes are observed and the duration of application of remineralizing agents is short than the recommended period.

Well designed & high-quality clinical studies in this area are still required before definitive recommendations. Hence many in vitro and invivo studies should be done with huge sample size to justify the results.

Conclusion

GC Tooth Mousse & SHY-NM are effective in remineralizing artificially induced caries lesions compared to Biomed calcimax and Bentodent .

The use of CPP–ACP caused in a substantial decrease in lesion depth associated with the positive and negative control, Biomed calcimax & Bentodent; conversely, it was statistically insignificant when compared to SHY NM. No significant difference was found between Group 3 and group 4; Group 4 and group 5; Group 5 and group 6. Henceforth CPP-ACP in GC Tooth Mousse is superior to SHY-NM, Biomed calcimax & Bentodent tooth paste.

Acknowledgments: None

Conflict of interest: None

Financial support: None

Ethics statement: The study was reviewed and approved by the Institutional Review Board (approval number 200/IHEC/1-19).