Mechanism of Action of a Desensitizing Fluoride Toothpaste Delivering Calcium and Phosphate Ingredients in the Treatment of Dental Hypersensitivity. Part II: Comparison With a Professional Treatment for Tooth Hypersensitivity

Andrew J. Charig, BS; Stephen Thong, PhD; Florita Flores, BS; Shivank Gupta, BS; Elizabeth Major, BS; and Anthony E. Winston, BSc

Dentin, the mineral organic layer underlying tooth enamel, is permeated with many tiny tubules, which provide passageways to the pulpal cavity where the intradental nerves are.¹ However, until about the third or fourth decade of life in healthy individuals, the surface of dentin is not exposed and the tubules are sealed.^2-5 With age, the gums often recede, exposing cementum, a thin layer of mineral covering the subgingival dentin.^2,3 When this layer is inevitably lost because of toothbrush abrasion or attack by acidic beverages and foods, the dentinal tubules are opened to the oral cavity and provide a direct connection to the intradental nerve.^2,3,5,6 Under such circumstances, individuals often experience pain caused by mild external stimuli, such as hot or cold foods and beverages.^2-5 This is because these stimuli change fluid flow within the open tubules, pressuring and activating the nerves.^1-8

A more serious cause of hypersensitivity can occur when there is access to the dentin because of cracking or loss of enamel from attrition, abrasion, erosion, caries, or other trauma.^2,4 The ramifications of these more serious problems may make superficial treatments ineffective and require more extensive intervention by a dentist.

Ishikawa et al evaluated in vitro the sequential application of an acidified solution of calcium phosphate, followed by a sodium hydroxide solution to the surface of the dentin, as a means of precipitating calcium phosphate to block open tubules and, hence, to treat hypersensitivity.⁹ The authors found plugs of dicalcium phosphate dihydrate (DCPD) had formed, which extended 15 µ into the tubules.

Using the same technique, Suge et al had shown the presence of fluoride resulted in the formation of plugs that were more apatitic in nature.¹⁰ Apatitic mineral is the least soluble calcium phosphate salt and closer than DCPD to the natural mineral present in dentin and tooth enamel.¹¹

The application of solutions that form amorphous calcium phosphates (ACP) on dentin was evaluated by Tung et al and Periera et al.^12,13 ACP forms rapidly when concentrated aqueous solutions containing calcium and phosphate ions are mixed. An advantage of ACP is that it coats dentin as a layer of tiny particles, which are extremely efficient at covering the tubules. Once formed, under the mouth’s moist conditions, ACP is unstable to reformation as a less soluble calcium phosphate salt. Under many conditions and particularly in the presence of a small quantity of fluoride ions, ACP reforms as a hard, coherent apatitic mineral similar in composition to tooth enamel and bonds strongly to the dentin to become an intrinsic part of the tooth.^14,15 Using this technique, Tung et al obtained dentin permeability reductions of 85% to 99.9% in the standard in vitro test, using coronal dentin in Pashley cells.^12,16

In a pilot study involving 16 participants with hypersensitivity from gum recession, four applications of concentrated calcium chloride solution followed by tripotassium phosphate solution applied once weekly to their sensitive teeth reduced thermal sensitivity by 72% (n = 11) compared to a reduction of 28% (n = 5) for a potassium chloride control group.¹⁷ Similarly, tactile sensitivity was reduced. One year after application, no significant sensitivity recurred. In a somewhat larger study (N = 30), Geiger et al confirmed the effectiveness of ACP-forming treatments compared to a placebo.¹⁸ This dual-solution technology has been commercialized as a desensitizing treatment for application in the dental office.

Adding a combination of calcium and phosphate salts to a fluoride toothpaste formulation normally would be problematic because of the potential formation of insoluble calcium fluoride and calcium phosphate, which would be inactive. However, to produce more effective home care products, toothpastes that simultaneously supply these ions have been developed. In these products, the calcium, phosphate, and fluoride sources are prevented by various means from reacting until exposed to saliva in the mouth. In vitro and in vivo studies have provided evidence that such toothpastes produce ACP on the enamel surface, filling in surface defects, smoothing the surface, and making the surface more glossy.^19-22

In an 8-week trial, a two-phase fluoride toothpaste employing silica abrasive and containing calcium sulfate and ammonium phosphate had been shown to alleviate tooth hypersensitivity more effectively than a conventional fluoride toothpaste, as demonstrated by the number of teeth that became insensitive by the trial’s conclusion.²³

Furthermore, a single-phase fluoride toothpaste (for treating hypersensitivity) that delivers calcium sulfate, dipotassium phosphate, and baking soda to the mouth from a non-aqueous carrier had been demonstrated in a controlled clinical study to provide highly effective relief from tooth hypersensitivity.²⁴ In addition, the reduction in hypersensitivity achieved with this toothpaste had been shown to be retained for at least 1 month after discontinuance of the desensitizing formulation.

In the first of this series of three articles, the authors discussed in vitro studies designed to elucidate the mechanism of action of the clinically proven fluoride toothpaste for treating hypersensitivity.²⁵ The results indicated when the toothpaste was applied to the teeth, the calcium and phosphate salts reacted on the dentin surface, depositing ACP. Because of the presence of moisture in the mouth, ACP transformed into a coherent layer of apatitic mineral on the surface of the dentin, thereby blocking the tubules.

The objective of the in vitro studies described in this article was to compare the likely mechanism of action of fluoride toothpastes delivering calcium and phosphate salts with that of the professional desensitizer in the treatment of hypersensitivity.

Materials and Methods

The products compared in these in vitro studies were:

• A professional desensitizer: Quell^® desensitizer (Q) (Pentron Clinical Technologies, LLC, www.pentron.com), which is applied in the dental office to treat hypersensitivity and consists of sequentially applied solutions of calcium chloride and tripotassium phosphate containing 1000-ppm fluoride. This product has been clinically proven to reduce dental hypersensitivity, and its mechanism of action has been established to be the initial formation of ACP on the dentinal tubules, blocking them.^12,13,16-18
• A two-phase fluoride dentifrice for home use: Enamelon^® toothpaste (E) (no longer marketed), which delivers calcium sulfate and ammonium phosphate to the tooth surface. This dentifrice has been proven to reduce hypersensitivity in an 8-week double-blind study.²³ Because this product is no longer commercially available, laboratory-prepared batches of the two phases were used in this study.
• A two-phase fluoride toothpaste for home use: Arm & Hammer^® Enamel Care^® (AHEC) (Church & Dwight Co, Inc, www.churchdwight.com) containing calcium sulfate, dipotassium phosphate, and baking soda.
• A single-phase fluoride toothpaste with baking soda: Arm & Hammer^® Enamel Care^® Sensitive (AHECS) (Church & Dwight Co, Inc) containing dipotassium phosphate and a higher level of calcium sulfate specifically designed for increased efficacy in treating hypersensitivity. In a controlled double-blind clinical study, this product has been proven to provide lasting relief of sensitivity.²⁴
• A conventional fluoride toothpaste: Crest^® Fluoride Anticavity Toothpaste (C) (Procter and Gamble, www.pg.com) containing hydrated silica abrasive. This was used as a control.

Coronal dentin disks that were approximately 300 µ to 500 µ in thickness were cut from intact human molars, using an IsoMet saw (BUEHLER Ltd, www.buehler.com). Any with enamel areas toward the center or with pulp cavity horns were rejected. The disks were honed to about 50-nm smoothness by hand-rubbing under water on CarbiMet^® disks (BUEHLER Ltd) successively with 600-, 800-, and 1200-mesh silicon carbide grit, followed by the use of a polishing cloth with a 50-nm diamond suspension. They were finally lightly etched with 5% citric acid for 15 secs to open the tubules. After mounting in Pashley cells, they were perfused with dye-colored, phosphate-buffered saline at pH 7.4 under a static head of about 75 cm of water (0.07 atm). The flow rate of liquid through the dentin was measured in µL/min. After several determinations of flow rate, the dentin was treated with the test products. After treatment, the flow rate was re-determined and the reduction in dentin permeability calculated. Because the purpose of these studies was to determine the product mechanism rather than to compare effectiveness, each test was duplicated and no efforts were made to establish statistical differences in permeability reduction performance.

For evaluation of the professional desensitizer, two drops of solution A were applied to a dentin disk mounted in a Pashley cell and the dentin sat for 30 secs. Two drops of solution B were then applied, and the combination was allowed to react for 60 secs. The dentin disk was rinsed in distilled water before re-determining the fluid flow rate.

The fluoride toothpastes were evaluated in duplicate for their ability to reduce the permeability of coronal dentin disks in Pashley cells. While held in the cell, the surface of each dentin disk was brushed for 5 mins with a freshly prepared 1:3 aqueous slurry of the toothpaste being tested, using a modified electric toothbrush (Interplak^®, CONAIR Inc, www.conair.com) with all but one set of tufts removed. About 50 g to 100 g of applied force was applied. The brushings were repeated multiple times, using fresh toothpaste slurry each time, and the dentin was rinsed between brushings with distilled water. Dentin permeability was determined after 5-, 10-, 15-, and 30-min cumulative treatment times.

After removal from the Pashley cells, scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analyses of the untreated, desensitizer-treated, and toothpaste-treated dentin surfaces were performed. The SEM and EDX equipment was a JEOL 6400F (JEOL Ltd, www.jeol.com) with INCAEnergy-dispersive analyzer (Oxford Instruments, www.oxinst.com), and a beam energy set from 5 kV to 15 kV.

Results

Figure 1 provides graphs of average dentin permeability reductions obtained through treatment with the professional desensitizer, the three fluoride toothpastes delivering calcium and phosphate salts, and the conventional fluoride toothpaste control. It can be seen that approximately 15 mins of cumulative treatment time by the calcium- and phosphate-containing toothpastes resulted in a similar dentin permeability reduction to that obtained with a single application of the professional desensitizer. Longer cumulative treatment times by the toothpastes further reduced dentin permeability. While the conventional toothpaste used in this study reduced dentin permeability, the reduction in flow appeared to be less than that for the calcium- and phosphate-containing toothpastes. The reduction in permeability did not exceed about 40% after 30 mins of cumulative treatment time.

Figure 2 provides SEM views of the surface of untreated dentin (Figure 2a) and a well-mineralized area of the dentin due to treatment with the professional desensitizer (Figure 2b). The surface was covered with a thick layer of highly coherent mineral, blocking the tubules. It may be noted that not all of the dentin treated with the professional desensitizer was well mineralized. Presumably, it was this lack of complete coverage that resulted in the dentin permeability not having been further reduced.

The SEM images in Figure 3 show close-up views of cross-sections of untreated (Figure 3a) and Q-treated (Figure 3b) dentin. The coherent nature of the 2-µ to 3-µ thick surface layer on this part of the treated dentin was clearly seen, and bonding of deposited mineral with the dentin surface was apparent.

The EDX elemental analysis of the professional desensitizer-treated surface is consistent with the presence of ACP or HAP together with potassium, phosphate, and chloride ions. Based on the mineral’s coherent nature, what is observed in the SEM seems to be HAP, formed from hydrolysis of ACP, which was likely to have been initially deposited. No protein was detectable in the surface as shown by the lack of carbon in the EDX. This demonstrated that the analyzed portion of the dentin surface was well covered by the deposited mineral.

Figure 4a, Figure 4b and Figure 4c provides SEMs of cross sections of the dentin, which was treated with each of the calcium- and phosphate-containing fluoride toothpastes—E, AHECS, and AHEC. Each figure shows mineral covering the dentin surface and blocking most of the tubules. The layer produced by AHECS looked very similar to that resulting from treatment of dentin with the professional desensitizer. Furthermore, it appeared to be strongly bonded to the dentin.

EDX analyses of the untreated-dentin disks used in these studies generally indicated a 1.5 atomic ratio of calcium to phosphorous. This is typical of calcium-depleted apatite found in mildly etched dentin. After treatment with the toothpastes, the calcium-to-phosphorous ratio in the analyzed surface ranged approximately from 1.7 to 1.85. This is slightly higher than that of the ratio in pure ACP or HAP, which is 1.66. It probably indicates the presence of unreacted potassium phosphate, which was not completely washed away when the disks were rinsed.

Figure 5 shows a dentin surface treated with a conventional fluoride toothpaste. The surface was covered with localized areas of what appeared to be a very fine amorphous tubule-obstructing substance. Figure 6a and Figure 6b provide cross-sectional views of untreated dentin and dentin treated with the conventional fluoride toothpaste. The treated dentin had a very thin layer of deposit above the tubules. EDX analysis of the C-treated dentin showed it to be well covered with silica particles, with no detectable dentin exposed (lack of carbon peak from protein). Apparently, there was more efficient coverage of the dentin surface by silica than was seen in the SEM figures.

Discussion

This study’s finding of calcium phosphate deposition by the professional desensitizer was similar to that found by Tung et al who determined sequential applications of calcium and phosphate solutions to dentin disks produced a layer of ACP and resulted in significant reductions of as much as 99.9% in dentin permeability.^12,16 However, examination of the SEMs indicated the deposit formed in the authors’ study was more crystalline than that formed on the dentin in studies by Tung et al. As previously mentioned, ACP is unstable in the presence of moisture and known to transform into other less soluble crystalline calcium phosphate salts. Fluoride reportedly catalyzes the transformation of ACP to apatitic mineral.¹⁰ Depending on how soon after treatment the SEM was obtained, ACP, HAP, or mixtures of the two could be present. In this study, it appeared the mineral had largely transformed into the more crystalline HAP mineral. The SEM of the dentin cross section shows a highly coherent nature and a strong bond with the dentin surface.

The calcium- and phosphate-delivering toothpastes apparently operate by a similar mechanism to the professional desensitizer, producing ACP that transforms into HAP on the surface of the dentin, sealing tubules and reducing dentin permeability. The morphology of the surface mineral produced by E and AHEC is not clearly seen in these SEMs. However, the mineral created by AHECS appeared strikingly similar to that made by Q. In both cases, the mineral appeared strongly bonded to the dentin surface.

In this study, the conventional toothpaste also deposited particles on the surface of the dentin and, as a result, exhibited some ability to reduce dentin permeability in the Pashley cell. Because of the test and control toothpastes used, no attempt was made to fully quantify differences in dentin permeability reduction. The reductions from the conventional toothpaste were numerically much less than those caused by the calcium- and phosphate-containing toothpastes. The deposit from the conventional toothpaste was silica—the insoluble abrasive employed in this toothpaste. Despite the drop in dentin permeability, conventional toothpastes are not generally considered to effect reductions in dentin hypersensitivity because of tubule occlusion in vivo. This contradiction was consistent with the findings in other studies. Thus, the presence of residues, which inhibit permeability because of the use of conventional toothpastes, has also been reported by others.^1,26-31

Pashley et al reported that the conventional fluoride toothpaste used in the current study actually reduced dentin permeability to a greater extent than two tubule-occluding desensitizing dentifrices they evaluated. While occlusion of dentinal tubules by inactive insoluble ingredients, such as silica abrasives, is not generally considered adequately effective for providing significant relief of hypersensitivity, it has been proposed as a possible reason why placebo dentifrices often perform better than expected in clinical trials.⁴ Others have even proposed the possible therapeutic potential of abrasives.^7,9

Markowitz et al and Pashley et al provided a reasonable explanation for the lack of in vivo efficacy of some insoluble toothpaste ingredients that are deposited in vitro and which reduce permeability in the Pashley cell.^32,33 Pashley et al found that, while a smear layer on dentin can reduce dentin permeability by 90% when measured hydrostatically, it only reduced permeability by 50% when measured using an air blast.³² They proposed that an air blast onto closely packed particles mixed with water can induce evaporation of some moisture and the surrounding fluid can be induced to flow through capillary action. Such flow cannot be induced hydrostatically. Presumably, evaporative flow can still be sensed by the intradental nerves.

When layered onto dentin, the insoluble abrasives left by a toothpaste do not bond with the dentin surface or form a coherent layer above the tubules. This leaves room for evaporative flow when an air blast is applied. While the SEMs in this study did not clearly show the particulate nature of the residue from the conventional toothpaste, the SEMs published in the authors’ previous article did demonstrate small particles of silica deposited by the control toothpaste used in that study.²⁵ Presumably, this difference was because of the variations in particle size of the adhering silica in each case. Importantly, while either type of insoluble silica particles may have the ability to interfere with dentinal flow in the Pashley cell, they presumably are significantly less effective in preventing evaporative flow with the more realistic pressures in the mouth. This evaporative type of flow may be sufficient to elicit pain.

All three fluoride toothpastes delivering calcium and phosphate salts appeared to result in the production of a coherent layer of mineral that was bound to the dentin surface. The mineral layer produced by AHECS and its attachment to the dentin appeared especially similar to that produced by the professional desensitizer. It is likely that the formation of this layer accounted for the efficacy of AHECS in effectively treating hypersensitivity.

Conclusions

The mechanism of action of the fluoride toothpastes that deliver calcium and phosphate ions to the tooth surface are the same as that of the two-part professional treatment, which uses sequentially applied concentrated solutions of calcium chloride and tripotassium phosphate containing sodium fluoride. Both products initially produce ACP on the dentin surface. ACP subsequently transforms into a coherent HAP layer bonded to the dentin. Sealing of the dentinal tubules with this tooth mineral-like layer reduces the permeability of fluids through the dentin, thereby blocking the effects of oral stimuli on the intradental nerves.

Disclosure

This research was supported by Church & Dwight Co, Inc. Mr. Charig was an employee of Church & Dwight, and Dr. Thong and Ms. Major are current employees. Dr. Thong is a shareholder of Church & Dwight. Mr. Winston is a former Church & Dwight employee. He is a shareholder and serves as a consultant for the company.

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About the Authors

Andrew J. Charig, BS
Senior Chemist (retired)
Church & Dwight Co, Inc
Princeton, New Jersey

Stephen Thong, PhD
Director
Global Oral Care R&D for Oral Care Products
Church & Dwight Co, Inc
Princeton, New Jersey

Florita Flores, BS
Technician (former)
Church & Dwight Co, Inc
Princeton, New Jersey

Shivank Gupta, BS
Technician (former)
Church & Dwight Co, Inc
Princeton, New Jersey

Elizabeth Major, BS
Consultant
Church & Dwight Co, Inc
Princeton, New Jersey

Anthony E. Winston, BSc
President
R&D for Hire LLC
East Brunswick, New Jersey