Dentin hypersensitivity is characterized by short and sharp pain in response to thermal, evaporative, tactile, osmotic, and chemical stimulations [1]. This is primarily due to exposure of the dentinal tubules caused by enamel loss and/or gingival recession. Several pathways have been proposed as mechanisms for pain sensitization of exposed dentinal tubules, and the hydrodynamic theory is the most widely accepted concept [2]. Hydrodynamic theory states that pain impulses are triggered by the movement of tubular contents or tubular fluid [3]. Therefore, dentin hypersensitivity is alleviated when fluid movement is inhibited by reducing dentin permeability.

The most common method to reduce dentin permeability is to occlude the exposed dentinal tubules through repeated treatments with chemical agents such as dentifrices and varnishes. Many products available in the market occlude the dentinal tubules. They mainly contain divalent metal ions such as Sn²⁺, Zn²⁺, and Sr²⁺ ions, or potassium salts such as potassium nitrate, potassium oxalate, and potassium phosphate as active ingredients. Notably, these ingredients can act to occlude dentinal tubules [4-6]. In addition, divalent metal ions can be incorporated below the surface of hydroxyapatite (HAp) to stabilize the apatite surfaces against acid erosion [7]. Potassium salts can penetrate open dentinal tubules and desensitize nerve fibers, ultimately reducing dentin hypersensitivity [8].

Although combined usage of metal ions and potassium salts to achieve synergistic effects is desirable, their application in pharmaceutical formulations is limited. These two components react in an aqueous solution to form an insoluble co-precipitate. To achieve an excellent dentin hypersensitivity reduction effect while ensuring the stability and effectiveness of the formulation, a method for stabilizing them in an aqueous solution is required. Recently studies have revealed that milk protein hydrolysates (MPHs) prevent precipitation caused by the reaction between metal ions and mineral components. Feng et al. [9] reported the formation of zinc/calcium phosphate/MPHs nanocomplexes stabilized in water. These nanocomplexes played a role in increasing the absorption of zinc ions in the small intestine.

Using MPHs, we present a method to stabilize ZnCl₂ and potassium phosphates, which are the active ingredients used for dentinal tubule occlusion. They spontaneously form nanocomplexes in aqueous solutions. In addition, the nanocomplexes rapidly aggregate when in contact with HAp, a tooth mineral. A dentifrice formulation containing the nanocomplexes was prepared, and its efficacy in occluding dentinal tubules was evaluated in vitro and compared with that of commercially available desensitizing dentifrices. We demonstrated the benefits of the nanocomplex-containing dentifrice by measuring dentin permeability after repeated application of dentifrice formulations to exposed dentinal tubules.

1. Preparation of MPH complexes

The MPH used was Lacprodan^® DI-2021 (Alra Foods Ingredients, Denmark), which is a partially hydrolyzed casein protein with a high content of casein phosphopeptides (CPPs). Zinc chloride was purchased from Merck (Darmstadt, Germany). Potassium dihydrogen phosphate (KH₂PO₄) and potassium hydrogen phosphate (K₂HPO₄) were purchased from SDBNI (Seoul, Korea). In all mixtures, MPHs were dispersed in distilled water (DW) to a final concentration of 0.5% (w/w). ZnCl₂, KH₂PO₄, and K₂HPO₄ were added to the mixture at concentrations of 0.09% (w/w), 2% (w/w), and 3% (w/w), respectively. ZnCl₂, KH₂PO₄, and K₂HPO₄ are the active ingredients of Sirin Takhyo Plus Toothpaste (LG H&H, Korea), which is used for reducing dentin hypersensitivity. The concentrations of ZnCl₂, KH₂PO₄, and K₂HPO₄ in the test were the same as those in the Sirin Takhyeo Plus Toothpaste. When a mixture containing MPHs, ZnCl₂, KH₂PO₄, and K₂HPO₄ was prepared, ZnCl₂ and MPHs were first mixed in DW, and then KH₂PO₄ and K₂HPO₄ were added.

2. Size measurement of MPH complexes

The size of the dispersion containing the MPH complexes was measured using a Zetasizer Nano-ZS90 (Malvern, UK). Particle diameters were assessed through dynamic light scattering measurements. The refractive index (RI) of DW, the dispersant, was set to 1.33 and the RI of the material was set to 1.45. The HAp used in the size measurement test was powdered and had a mean diameter of 150 nm. The HAp powder was added to solutions containing the MPH complexes to a final concentration of 0.1% (w/w), and the solution was vigorously shaken. To monitor the size change of the MPH complexes due to the addition of HAp powder, the mean diameter was measured at 0, 1, 2, and 3 minutes after addition.

3. Preparation of dentifrices

A dentifrice prototype was prepared containing pre-mixed complexes of ZnCl₂, MPHs, KH₂PO₄, and K₂HPO₄ to the same base formulation as the Sirin Takhyo Plus Toothpaste. The final concentrations of ZnCl₂, MPHs, KH₂PO₄, and K₂HPO₄ in the toothpaste were 0.09% (w/w), 0.5% (w/w), 2% (w/w), and 3% (w/w), respectively. Additionally, desensitizing toothpastes available in the market, including Sirin Takhyeo Plus Toothpaste, were used for testing. The toothpastes used in the test are representative desensitizing toothpastes from global companies, and are the most easily accessible to patients with dentin hypersensitivity. The toothpaste samples were selected based on criteria such as sales orders and actual accessibility.

The key active ingredients of the dentifrices used in the tests are presented. Among the active ingredients of dentifrices, only those related to dentinal tubule occlusion were selected and are shown as key active ingredients. Therefore, in addition to the key active ingredients, all dentifrices contain other active ingredients such as abrasives and anti-gingivitis agents; however, these are not shown in the table to clearly present the variables that affect dentinal tubule occlusion. To be precise, the prototype dentifrice used in the actual test contained the following active ingredients: 1.1% sodium monofluorophosphate (SMFP; anti-cavity agent), 10% dental-type silica (abrasive), 10% calcium carbonate (abrasive), 0.01% tocopheryl acetate (anti-gingivitis agent), 0.02% Zea mays L. extract (anti-gingivitis agent), 0.01% Centella asiatica extract (anti-gingivitis agent), 0.05% magnolia bark extract (anti-gingivitis agent), 2% monobasic potassium phosphate (desensitizing agent), 3% dibasic potassium phosphate (desensitizing agent), and 0.09% zinc chloride (desensitizing agent).

4. Assessment of dentin permeability

First, dentin specimens were prepared with the dentinal tubules in the vertical direction. The dentin of bovine teeth was cut flat and polished with silicone sandpaper to produce disc with a thickness of 500 μm. The disc was masked with polyimide film (# 5413 K, 3M) to ensure that only an area of approximately 1 cm×0.5 cm dentin was exposed to the outside. The discs were then immersed in 0.5 M ethylenediaminetetraacetic acid (EDTA) solution (pH 7.5) for 1 hour to completely expose the dentinal tubules. The discs were dried and stored at room temperature. The disc that had been stored was re-immersed in 0.5 M EDTA solution for 10 minutes and thoroughly washed with DW before use.

Toothpaste samples were diluted 1:3 with artificial saliva to form a slurry and applied to the dentin specimens. The toothpaste slurry was applied for 1 minute. After the treatments, the specimens were completely washed with tap water and stored in artificial saliva at 37°C. Toothpaste treatment was repeated twice a day for a total of six times over three days. Under conditions that induce erosion by acids, the process of treatment with toothpaste slurry for 1 minute, washing with tap water, treatment with 0.01 M citric acid solution (pH 2.5) for 1 minute, and washing with tap water was repeated six times.

The permeability of dentin was confirmed by measuring the force required for artificial saliva to pass through the dentinal tubules. The masked dentin specimen was firmly mounted between the O-rings on an extruder (Avanti mini-extruder; Avanti Polar Lipids, USA). When artificial saliva was injected using a syringe from one side of the extruder, it passed through the dentinal tubules of the dentin specimens and flowed out on the other side. The force applied to the syringe was monitored using a TA-XT plus texture analyzer (Stable Micro Systems, England). The test speed for pressing the syringe was set at 0.2 mm/s. The forces measured before and after toothpaste treatment are denoted as F_i and F_a, respectively. The rate of change in the force is expressed as F_a/F_i, which indicates the level of reduction in dentin permeability. The statistical significance (p<0.05) of the differences in the F_a/F_i values obtained from each toothpaste treatment was verified using one-way analysis of variance (ANOVA) with Tukey’s HSD test.

The ability of the MPHs to form complexes with ionic salts was confirmed by monitoring the size of the dispersions (Figure 1). The MPHs were fully swollen when dispersed in DW and their mean diameter was approximately 433 nm. When 0.5% MPHs were dispersed in an aqueous solution of 0.09% ZnCl₂, the mean diameter of the reactants increased significantly to 1,187 nm. MPHs contained CPPs and had a negative surface charge of −25.3 mV (data not shown). Therefore, the MPHs were associated with the mediation of Zn²⁺ ions, resulting in a notable increase in the size of the dispersions. No change in the mean diameter was observed when the MPHs were mixed with 5% potassium phosphates. However, when ZnCl₂, MPHs, and potassium phosphates were mixed together in DW, the mean diameter was 556 nm, which was slightly larger than that of the MPHs.

Figure 1. Size distribution of MPHs and MPH complexes containing Zn²⁺ ions and potassium phosphates. MPHs were dispersed in DW at a concentration of 0.5% (w/w). The concentrations of ZnCl₂, KH₂PO₄ and K₂HPO₄ were 0.09% (w/w), 2% (w/w) and 3% (w/w), respectively.

To understand the characteristics of the MPHs and MPH complexes upon contact with HAp, the effect of the contact time on the change in their size was observed. As shown in the size distribution diagrams in Figure 2, although the HAp powder with a diameter of 150 nm was dispersed, no particles of this size were observed in the graphs. This is because HAp can bind to MPHs owing to the calcium-binding behavior of CPPs, which are the main components of MPHs [10]. The sizes of the dispersions were measured at 0, 1, 2, and 3 minutes after the addition of the HAp powder, and the characteristics of the size change were monitored. The MPHs showed a slight increase in the mean diameter when bound to HAp, but did not change with contact time (Figure 2A). The complexes of MPHs and ZnCl₂ had a mean diameter of 1,187 nm (Figure 1). However, with the addition of HAp, the mean diameter decreased to approximately 500 nm and did not change with the contact time (Figure 2B). For the complexes of MPHs and potassium phosphates, the diameters increased slightly depending on the contact time (Figure 2C). The complexes of ZnCl₂, MPHs and potassium phosphates exhibited a rapid increase in size upon contact with HAp. When the contact time reached 3 min, the mean diameter of the complexes increased from 561 nm to 906 nm. This is because the complexes aggregate owing to a decrease in the zeta potential.

Figure 2. Size distribution graphs showing the diameter change of (A) MPHs, (B) MPHs with ZnCl₂, (C) MPHs with potassium phosphates, and (D) MPHs with ZnCl₂ and potassium phosphates when they contact HAp. HAp is in powder form with a mean diameter of 150 nm and is dispersed in DW at a concentration of 0.1% (w/w).

The effect of a prototype dentifrice containing complexes of ZnCl₂, MPHs and potassium phosphates on occluding dentinal tubules was confirmed. In the test, dentifrices were diluted 1:3 with artificial saliva and dentin specimens were soaked in the slurry for 1 minute. The dentin specimens have exposed dentinal tubules that allow fluid to move through them, and the force required for the fluid to penetrate them has been measured. Because occlusion of the dentinal tubules directly limits the movement of fluid, efficacy was quantitatively assessed by comparing fluid permeability before and after treatment with dentifrices.

The dentin specimens were treated with the test dentifrices six times in slurry form, and changes in permeability were measured. Dentifrices, whose active ingredients consisted of a combination of SnF₂, SMFP, KNO₃, ZnCl₂, and potassium phosphates, increased the force required to move through the tubules increased 1.32-1.57 times after treatments with them (Table 1). For the dentifrice containing the complexes of ZnCl₂, MPHs and potassium phosphates, a 1.78-fold increase in force was observed (Table 1). A statistically significant difference was observed between the results of the prototype and those of the other six dentifrices.

Table 1 . Changes in the force required to move fluid through dentinal tubules by repeated treatments with dentifrices

	Key active ingredients	Sample	Fa/Fi
Commercial dentifrices	SnF2	A1	1.35±0.15
		A2	1.45±0.21
	SMFP	B	1.35±0.11
	SMFP, KNO3	C1	1.45±0.05
		C2	1.32±0.05
	SMFP, ZnCl2, KH2PO4, K2HPO4	D	1.57±0.16
Prototype	SMFP, ZnCl2, MPHs, KH2PO4, K2HPO4	E	1.78±0.14

Acid resistance was tested by repeatedly inducing acid erosion in dentin specimens through exposure to 0.01 M citric acid for 1 minute after treatment with dentifrices. Among the commercial dentifrices, those containing SnF₂ as an active ingredient showed excellent acid resistance. The differences between the SnF₂ dentifrice groups (Table 2) and the other dentifrice groups (Table 2) were significant. The dentifrices containing the complex of ZnCl₂, MPHs and potassium phosphates (Table 2) showed the highest acid resistance at a statistically significant level compared with commercially available dentifrices, including the SnF₂ group.

Table 2 . Changes in the force required to move fluid through dentinal tubules by repeated treatments with dentifrices and acid

	Key active ingredients	Sample	Fa/Fi
Commercial dentifrices	SnF2	A1	1.29±0.11
		A2	1.31±0.18
	SMFP	B	1.04±0.18
	SMFP, KNO3	C1	1.04±0.18
		C2	1.15±0.18
	SMFP, ZnCl2, KH2PO4, K2HPO4	D	1.11±0.16
Prototype	SMFP, ZnCl2, MPHs, KH2PO4, K2HPO4	E	1.54±0.17

CPPs, the main components of MHPs, are good carriers and solubilizers of mineral components [11]. In particular, CPPs combine with amorphous calcium phosphates (ACPs) to form a CPP-ACP complex, which helps remineralization of tooth enamel [12]. We used potassium phosphates instead of ACPs to retain the additional benefits of potassium ions in reducing dentin hypersensitivity. Many reports have demonstrated the desensitizing effect of potassium-containing dentifrices [13], and it has been postulated that potassium ions released from dentifrices diffuse through the dentinal tubules and deactivate intradental nerves [14]. Although no results regarding the effects of complexes of MPHs and potassium phosphates have been reported, owing to the chemical similarity between calcium phosphates and potassium phosphates, a complex of MPHs and potassium phosphates similar to that of CPP-ACP may be formed. As shown in Figure 2C, dispersions composed of MPHs and potassium phosphates also increased in size when in contact with HAp. Because CPP-ACP occludes the dentinal tubules [15], a complex of MPHs and potassium phosphates is expected to have a similar effect.

However, providing acid resistance to dentin using simple mineral occlusion is difficult. Therefore, we used Zn²⁺ ions in combination with MPHs and potassium phosphates. Zinc is a key trace element in oral care. Zn²⁺ ions have been proven effective in treating or preventing common oral health problems such as cavities, gum inflammation, and oral malodor [16]. It also inhibited collagen degradation in acid-etched dentin [17]. The effect of zinc that we focused on is that it adsorbs to HAp and stabilizes HAp against acid attack [7]. Sn²⁺ ions have the same effect as Zn²⁺ ions. The interaction of HAp with Sn²⁺ ions released from SnF₂ dentifrices has been shown to reduce HAp solubility by up to 80% compared to NaF dentifrices [18]. Owing to these characteristics of divalent metal ions, when the dentinal tubules were occluded with SnF₂ dentifrices, the occlusion showed resistance to acids (Table 2). Divalent metal ions adsorbed on the HAp surface can be incorporated below the surface by substitution with Ca²⁺ ions. Because Zn²⁺ ions are better adsorbed and incorporated into hydroxyapatite than Sn²⁺ ions [7], Zn²⁺ ions were chosen for use in this study, and excellent acid resistance was achieved. In addition, components such as SnCl₂ as well as ZnCl₂ can effectively exhibit acid resistance. Further research is required to determine the synergistic or competitive effects of these two ingredients. The interaction of ZnCl₂, calcium salts and MPHs has been reported, and studied for their application as a supplementary nutrient [9]. We used potassium salts instead of calcium salts to induce additional desensitization of nerves in the oral cavity, and it is significant that a new application area of alleviation of dentin hypersensitivity has been revealed.

To optimize the effectiveness of Zn²⁺ ions, their ionic properties must be maintained when delivered to the dentin surface. Zn²⁺ ions can react with phosphates, which are widely present in pharmaceutical formulations and in the oral cavity, to form an insoluble precipitate of Zn₃(PO₄)₂. Zinc phosphate can be utilized in dentistry as a joint material between teeth and crowns because of its high fracture toughness, low solubility, and high bonding strength with adhesives [19-21]. However, for the general daily use of chemical agents, factors such as short usage time and comfort during use are also very important. Therefore, developing daily use agents in which zinc phosphate exhibits bonding properties without other adhesives is difficult. Therefore, to stabilize the Zn²⁺ ions, a strategy for forming a complex with MPHs and potassium phosphates was adopted. More specifically, minimizing the formation of zinc phosphates is required. Thus, as described in the Methods section, ZnCl₂ and MPHs were first mixed and then potassium phosphates were added. The order of mixing is important for suppressing the formation of precipitates, and the reaction between Zn²⁺ ions and phosphates should be avoided. Through this process, we stabilized the Zn²⁺ ions by protecting them from phosphates, resulting in dentinal tubule occlusion and the high acid resistance of dentin minerals.

In this study, the degree of occlusion of the dentinal tubules was quantified based on the force applied when the fluid moved through the dentinal tubules. The complex of ZnCl₂, MPHs, and potassium phosphates effectively occluded the dentinal tubules. As the size of the complex of ZnCl₂, MPHs, and potassium phosphates increased depending on the contact time with HAp, it could be inferred that occlusion was induced because they bound to the dentin surface and aggregated. The estimated mechanism is schematically shown in Figure 3. Because MPHs bind calcium, the complex of ZnCl₂, MPHs, and potassium phosphates are adsorbed onto the dentin surface. Subsequently, the complex and dentin surface are integrated through the action of Zn²⁺ ions, and the complexes aggregate and gradually occlude the exposed dentinal tubules. This hypothesis can be more clearly proven through additional studies analyzing the Zn²⁺ ions remaining on the dentin surface after toothpaste treatment. Furthermore, in this study, a prototype was formulated with fixed contents of active ingredients based on those of Sirin Takhyo Plus Toothpaste. However, for better efficacy, it is necessary to evaluate the interaction between active ingredients and the effect of the combination of contents.

Figure 3. A schematic diagram showing the principle of dentinal tubule occlusion by the complex of ZnCl₂, MPHs, potassium phosphates.

This study demonstrated the efficacy of a dentifrice containing the complex of ZnCl₂, MPHs, and potassium phosphates in occluding dentinal tubules. ZnCl₂, MPHs and potassium phosphates formed complexes in aqueous solutions. When the complex came into contact with HAp, the main mineral component of teeth, self-aggregation was promoted. The complex of ZnCl₂, MPHs, and potassium phosphates was then used as a new dentifrice formulation. Repeated treatment of the dentin surface with the developed dentifrice resulted in dentinal tubule occlusion. Dentinal tubule occlusion was demonstrated by quantifying the force required for fluid to penetrate the dentin. The developed dentifrice was not only effective in dentinal tubule occlusion but also provided high acid resistance to dentin. The strategy of using MPHs to stabilize ZnCl₂ and potassium phosphates to induce dentinal tubule occlusion can be effectively utilized in the development of a new toothpaste to alleviate dentin hypersensitivity.

No potential conflict of interest relevant to this article was reported.