Bioactive remineralization of dentin surface with calcium phosphate-based agents: An in vitro analysis.

Background: With the increasing prevalence of erosive tooth wear affecting both adults and children; designing optimum protocols of management in a noninvasive manner is gaining precedence. Aim: Comparative evaluation of topically applied calcium phosphate-based agents casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and beta tricalcium phosphate (β-TCP) on the surface of eroded dentin. Materials and
Methods: Dentin blocks from human third molars in four groups were subjected to the surface treatment: (G1) sound dentin (G2) demineralized dentin (G3) CPP-ACP (G4) β-TCP. All the samples except control (G1) were immersed in an acidic solution incubated at 37°C for 96 h. The samples in G3 and G4 were topically treated with CPP-ACP and β-TCP for 4 min twice daily for 14 days; followed by pH-cycling for 21 days. Surface hardness testing and surface morphology were observed using the scanning electron microscopy. Data were analyzed using the Statistical Package for the Social Sciences (SPSS) software with Kruskal-Wallis test and post hoc test.
Results: Dentin treated with both CPP-ACP (37.25) and β-TCP (32.05) recorded significantly higher VHN than demineralized (G2-23.51) but lower compared to sound control (G1-57.06).
Conclusion: The topical application of CPP-ACP and β-TCP agents shows definite potential in promoting the hardening of demineralized dentin surface. Copyright:

INTRODUCTION

Mineralized tissues of teeth are continuously subjected to a dynamic balance of demineralization and remineralization due to constant fluctuations of temperature, moisture, the presence of microbial biofilm, and pH changes throughout the life in the oral cavity. When imbalanced, the demineralization process becomes dominant which chiefly occurs due to acidic challenges resulting from both caries and erosive processes. However, in the carious process, the rate of demineralization occurs faster than remineralization due to a state of biofilm dysbiosis caused by cariogenic bacteria, leading to the progressive breakdown of the enamel surface and exposure of underlying dentin.[12]

Dental caries is a highly prevalent chronic multifactorial transmissible infection that results in the dissolution of tooth structure by acid metabolites from the bacterial fermentation of carbohydrates in the biofilm with its progress or reversal being determined by the balance between the pathological and the protective factors. In contrast, dental erosion is considered to be a surface phenomenon caused by exposure to the acid of nonbacterial origins[3] which advances by progressive loss of softened surface layers under further erosive-abrasive challenges.

As the traditional approaches of restorative management have demonstrated an annual failure rate of up to 7.9% mainly due to secondary caries at the marginal tooth-restoration interface and hypersensitivity related to erosion; contemporary approaches such as noninvasive intervention for induction of dentin surface remineralization in noncavitated lesions by therapeutic agents need to be explored for more effective management.[4] Traditional fluoride based and the newest age remineralizing agents primarily designed for remineralization of enamel have demonstrated successful results in previous studies.[5] Although the remineralization potential of fluoride on enamel is remarkable, it was found to be less effective on dentin.

Calcium phosphate-based bioactive agents such as casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) complex (GC, Tooth Mousse) and beta tricalcium phosphate (β-TCP) based dentrifice (3M ESPE, Clinpro Tooth Crème) primarily developed for their anti-cariogenic prophylactic effects have also been considered additionally in the management of dentin sensitivity following in-office procedures such as bleaching, scaling, and root planing. CPP-ACP complex (Tooth Mousse, GC Int., Japan) has shown promising results on dentin[6] while the addition of TCP to a dentifrice enhanced its ability in reducing dentine sensitivity.[7] Thus, this in vitro study was designed to evaluate and compare topical efficacy of CPP-ACP and β-TCP on the hardness and surface changes in the morphology of demineralized dentin.

MATERIALS AND METHODS

Specimen preparation

Six human third molars extracted from patients in the age group of 15–30 years were collected, screened for defects, and processed as per in vitro ethical research protocols and OSHA-CDC guidelines. To expose the dentin surface, the outer enamel of all teeth was eliminated using a water-cooled high-speed coarse-grit diamond point; sectioned with a low-speed diamond disc (SHOFU, Japan), horizontally about 2 mm below the CEJ and longitudinally into two buccal and lingual blocks each (approximately 4 mm × 4 mm × 2 mm). The four dentin blocks harvested from each molar were allocated into four groups of six samples, respectively, designated according to the surface treatment to follow: G1 (Sound Dentin-Control), G2 (DM-Demineralized), G3 (CPP-ACP), and G4 (β-TCP). The flat outer test area surface was finished with fine-grit diamond points and polished with silicon carbide abrasive paper (600–1200 grit) underwater cooling followed by the application of acid-resistant nail polish on all remaining sides.

Demineralization-remineralization regimen

Demineralization

For induction of surface demineralization that simulates a high erosive-cariogenic challenge all dentin blocks except G1 were immersed in an acetate-buffer based demineralizing solution (NaH2PO4:2.2 mM, CaCl2:2.2 mM, acetic acid: 0.05 M, NaOH: 50% and 0.5 ppm fluoride as NaF; pH 4.5) for 96 h at 37°C.[8]

Remineralization

Dentin surface of G3 and G4 was treated with CPP-ACP and β-TCP agents for 4 min twice daily for 14 days to simulate normal recommended daily tooth brushing. During the interim period, all group samples were stored in freshly prepared Artificial saliva of pH-7.2 [KCl-17.98 mM, NaCl2-4.29 mM, Na3PO4-3.90 mM, NaHCO3-3.27 mM, CaCl2-1.10 mM, H2SO4-0.50 mM, MgCl2-0.08 mM in distilled water] which was replaced every 24 h.

pH-cycling

For simulation of oral dynamics of mineral saturation and associated pH changes, samples were subjected to a pH-cycling based on reversal (Remineralizing) model[9] by alternative immersion in acid buffer (50 mM acetate, 2.25 mM CaCl2.H2O; 1.3 mM KH2PO4;130 mM KCl; pH-4.5) for 2 cycles of 1 h/day and remaining 22 h in artificial saliva (pH-7.2) for 21 days. Specimens were washed with de-ionized water for 2 min, blot dried, and stored under relative humidity at 37°C for 24 h before testing.

Surface microhardness test

Specimens mounted on acrylic blocks were placed on a Vickers Hardness Tester (Matsuzawa. Co. Ltd, Japan) to receive a series of five indentations from a diamond indenter perpendicular to the flat surface spaced 100 μm from each other under a load of 50 g for 15 s dwell time.[9] The diagonals of the indentation were imaged, measured under an optical microscope to calculate the average Vickers hardness number (VHN).[10]

Scanning electron microscopy examinations

Two dentin slabs per group were subjected to scanning electron microscopy (SEM, ZEISS) surface morphological evaluation to obtain representative photomicrographs of 20 μm at 10.00 kV under ×1000 magnification.

Statistical analysis

Table 1 shows the VHN data analysis for the mean, standard deviation, and nonparametric Kruskal-Wallis test for the overall comparison between the groups. Table 2 shows the post hoc test-Independent samples Kruskal-Wallis test for pair-wise multiple comparisons done using IBM Corp. Released 2015. IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY: IBM Corp.

Table 1

Vickers surface hardness measurements-mean and standard deviation

Group	Surface treatment	Mean VHN	n	SD	P
G1	Sound dentin	57.067	6	1.3411	0.001*
G2	DM	23.517	6	1.4593
G3	DM+CPP-ACP	37.250	6	1.5017
G4	DM + β-TCP	32.183	6	1.3920
	Total	37.504	24	12.6507

VHN: Vickers hardness number, SD: Standard deviation, β-TCP: Beta tricalcium phosphate, DM: Demineralized, CPP-ACP: Casein phosphopeptide-amorphous calcium phosphate, *Statistically significant when P < 0.05

Table 2

Pair-wise intergroup comparison (Kruskal-Wallis test)

Group	Test statistic	Standard test statistic	P	Statistical significance
G1 (sound)
G2 (DM)	−18	−4.409	0.001	S
G3 (CPP-ACP)	−6	−1.470	0.850	NS
G4 (β-TCP)	−12	−2.939	0.020*	S
G2 (DM)
G3 (CPP-ACP)	−12	−2.939	0.020*	S
G4 (β-TCP)	−18	−4.409	0.850	NS
G3 (CPP-ACP)
G4 (β-TCP)	−6	−1.470	0.850	NS

β-TCP: Beta tricalcium phosphate, DM: Demineralized, CPP-ACP: Casein phosphopeptide-amorphous calcium phosphate, S: Significant, NS: Not significant, *Statistically significant when P < 0.05

RESULTS

Surface microhardness (Vickers hardness number) measurements

The mean VHN measurements [Table 1 and Figure 1] were the highest for G1 (S: 57.06) followed by G3 (CPP-ACP: 37.25), G4 (β-TCP: 32.05) and least for G2 (DM: 23.51) respectively; were statistically significant (when P < 0.05). In inter-group comparison, [Table 2] statistically significant difference (when P < 0.05) seen between G1 and all groups except G3 (CPP-ACP), but not between G3 (CPP-ACP) and G4 (β-TCP).

Figure 1

Intergroup comparison of mean Vickers hardness number values

Scanning Electron Microscopy [SEM] Analysis: SEM Photomicrographs of Dentin surface at X1000 magnification shows smear layer covering the orifices of dentinal tubule in G1(S) - Sound Dentin [Figure 2a] which was completely removed exposing wider patent orifices in samples of G2(DM) [Figure 2b]. The precipitation and deposition of mineral particles with higher degree of occlusion of tubule orifices in G3(DM+CPP-ACP) [Figure 2c] compared to G4(DM+ b-TCP) [Figure 2d].

Figure 2

SEM Photomicrographs of Dentin surface at X1000 magnification (a) G1(S) -Sound Dentin –Smear layer on surface covering the orifices of dentinal tubule. (b) G2(DM) -Demineralized Dentin- Removal of smear layer and smear plug with greater number of wider patent orifices of dentinal tubules than G1. (c) G3(DM+CPP-ACP)- Precipitation and deposition of mineral particles with higher degree of occlusion of tubule orifices compared to G4. (d) G4(DM+β- TCP) Precipitation and deposition of mineral particles with relatively lower degree of occlusion of tubule orifices compared to G3

DISCUSSION

In a healthy mouth, the dentin surface is usually protected by the overlying enamel/cementum except in case of erosion, attrition, abrasion, and gingival recession is especially seen in the aging population. Despite protective factors such as salivary components and minerals to aid in remineralization; erosive-cariogenic acidic challenge in the presence of reduced salivary function can result in progressive surface demineralization decreasing the tooth hardness[11] with associated hypersensitivity and further erosive-abrasive surface loss ultimately causing pulpal exposure

With reports of the increasing prevalence of erosive tooth wear affecting both adults and children due to lifestyle change owing to excessive consumption of low-pH soft drinks,[12] the use of appropriate mineralizing agents is recommended for tooth-root surface protection to delay the need for restorative interventions, especially for patients in high-risk categories.[13] CPP-ACP and β-TCP, two of the advanced calcium phosphate-based bioactive products recommended for biomimetic remineralization of noncavitated lesions[14] tested in this study showed improvement in surface hardness of demineralized dentin, demonstrated mineral precipitation and occlusion of dentin tubules under SEM observation

Although dentin treated with CPP-ACP (37.25) showed relatively higher surface hardness compared to b-TCP (32.05), it was statistically not significant and attributed to the ability of CPP to stabilize localize ACP buffer reservoir in a state of supersaturation.[615] CPP ACP (GC Tooth Mousse) is a non-fluoridated, non-crystalline casein phosphopeptide (derived from casein milk protein) stabilized ACP complex that binds to plaque, hydroxyapatite; both localizing and increasing the bio availability of calcium, phosphate, and fluoride which has shown to significantly increase microhardness in incipient enamel and root surface caries lesions.[1617]

β-TCP (3M ESPE, Clinpro Tooth Crème) is a fluoridated (950 ppm, 0.21% w/w Sodium Fluoride) crystalline hybrid bioactive material manufactured by mechanochemical ball milling process.[9] This results in a functionalized tricalcium phosphate structure covered with a surfactant sodium lauryl sulfate to protect the bioavailable calcium from combining with fluoride ions in the paste; which breaks down on contact with saliva on application releasing calcium, phosphate, and fluoride ions.[16]

Amongst the control groups, the highest VHN values were noted in the G1-Sound dentin (57.067)[17] and lowest in the G2-Demineralized dentin (23.517)[18] which was statistically significant. When compared to enamel, the plate-shaped hydroxyapatite crystals of dentin are much smaller, poor in calcium but rich in carbon[8] with a higher rate of acidic dissolution due to its greater permeability. In this study, artificial demineralized erosive lesions were created according to Ten Cate and Duijsters, and a pH-cycling model[19] to simulate the dynamic oral environmental variations with artificial saliva as a storage medium to mimic the role of biological factors in the development of erosive lesions.

When the inorganic contents of tooth surface are lost by exposure to acid, decreasing tooth hardness[20] remineralization represents a biomimetic repair mechanism to promote ionic deposition of key mineral elements such as calcium, phosphate, and fluoride in the spaces of the crystal lattice.[14] In comparison to enamel, the thermodynamics of the demineralization/remineralization process in dentin is unique due to smaller dentinal crystals, more reactive associated pulp reactions of reparative dentin/sclerosis, a greater percentage of the organic matrix with complex structured phosphoproteins and water.[2122] Thus, continuous delivery and deposition of inorganic mineral alone is insufficient to restore mechanical properties of dentin; it is essential to preserve and stabilize the collagen matrix to serve as a template for mineral deposition by specific bioactive agents.[23]

Since the main interactions in the initiation and progression of both caries and erosion are predominantly surface phenomena, evaluations of changes in surface microhardness (SMH) are an important parameter in studying the demineralization-remineralization processes.[24] SMH measurement technique is a relatively simple, rapid, and nondestructive tool for a preliminary assessment in studies on demineralization-remineralization models.[21] Microhardness is resistance to the local deformation which measures the permanent surface deformation caused by an indenting stylus after the load removal. Dentin microhardness is highly variable due to its heterogeneous composition, microstructure, and sensitive toward the change of composition due to loss of inorganic components; hence, assessment of SMH can provide the initial step to determine the effect of mineralization protocols. Since microhardness changes in dentin may get influenced by shrinkage of the indentation due to elastic deformation[19] the VHN data in this study were recorded immediately after the indentation was performed.

The SEM analysis of dentin surface morphological features provided ultrastructural images of dentinal tubules covered with smear layer in G1 (S) [Figure 2a], while G2 (DM) [Figure 2b] showed complete removal of smear layer and smear plug with wide patent orifices of dentinal tubules. While both G3 (DM+CPP ACP) [Fig 2c] and G4 (DM+ b-TCP) [Figure 2d] showed precipitation and deposition of mineral particles with variable thickness in comparison with G2; CPP ACP in G3 showed a higher degree of dentinal tubule occlusion compared to G4 (b-TCP) which is in agreement with Oshiro et al.[25]

Bioactive remineralization of demineralized dentin surface can impart the following clinical benefits: Acid Resistance (Carious and Erosive challenges), Abrasion Resistance (Mechanical/Masticatory Wear), and Surface impermeability (Microleakage and hypersensitivity). Dentists have a professional responsibility in preserving the long-term health of teeth in potential individuals at risk by designing noninvasive conservative biomineralization protocols that include the application of protective toothpaste and creams over other complex restorative approaches in addition to dietary-lifestyle management.[20]

CONCLUSION

Based on the comparison of surface treatment modalities tested and study limitations, we conclude that while topical application of both agents displayed significant remineralizing potential; CPP-ACP showed relatively higher efficacy than β-TCP, but may require other additional strategies to stabilize organic collagen component for optimal improvement in mechanical properties to match sound dentin.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.