Synergistic efficacy of a biomimetic hydroxyapatite-citrate complex for dentin hypersensitivity: an in vitro and clinical study

Abstract

Background

To evaluate the synergistic effect of a novel Hydroxyapatite (HAP)-Citrate complex toothpaste on dentinal tubule occlusion and the relief of dentine hypersensitivity (DH).

Methods

For the in vitro study, thirty bovine dentin discs were randomly divided into three groups with ten specimens each: group A, a test toothpaste (HAP, potassium citrate and NaF), group B, a control toothpaste (HAP and NaF), and group C, a placebo toothpaste (NaF only). After 3 and 7 days of simulated brushing, tubule occlusion and mineralized layer formation was assessed using Scanning electron microscopy (SEM). A 12-week, double-blind, randomized trial involving 129 subjects compared the test toothpaste to a bioactive glass-based positive control toothpaste and a placebo. The severity of DH, indicated by Schiff (air) and Yeaple (tactile), was evaluated at baseline, 2, 6, 8, and 12 weeks, with the last 4 weeks being a washout period. Data were analyzed using ANOVA to compare differences between groups, with a significance level set at p < 0.05.

Results

In the in-vitro study, the HAP-Citrate complex toothpaste demonstrated rapid and effective occlusion. After 3 and 7 days, it achieved a dentinal tubule occlusion rate of 96.0 ± 2.1% and 99.7 ± 0.7%, significantly surpassing the placebo group (43.5 ± 10.1% and 76.4 ± 7.3%, respectively; p < 0.05). Notably, the HAP-Citrate complex exhibited superior deep-sealing capabilities compared to the standard HAP control toothpaste. At day 7, the HAP-Citrate group showed an intratubular occlusion depth of more than 40 μm (vs. 13 μm in the HAP control) and formed a surface mineralized layer of more than 5 μm thick (vs. 1 μm in the HAP control). In the clinical trial, the HAP-citrate toothpaste showed significant DH relief, with Schiff and Yeaple score improvements of 21.39% and 100.85% respectively at 8 weeks compared to placebo (p < 0.05), comparable to the positive control. Notably, the reduction in air-stimulus sensitivity was sustained at the 12-week follow-up, four weeks after the subjects discontinued the product use.

Conclusions

The novel HAP-citrate complex toothpaste can build deep, protective mineral seal on dentin tubules. Its clinically proven performance makes it a superior, science-backed choice for effective sensitivity management.

Registry

Chinese Clinical Trial Registry, TRN: ChiCTR2500115399, Registration date: 25 December 2025.

Background

Dentine hypersensitivity (DH) is characterized by short and sharp pain arising from exposed dentin in response to thermal, tactile, chemical, or osmotic stimuli, and cannot be attributed to dental defects or pathological changes caused by other specific factors [1]. DH is a common and frequently encountered dental problem. According to a published result of random-effects meta-analyses in 2019, the incidence rate of DH is about 33.5% on average, with high heterogeneity ranging from 1.3% to 92.1%, because of differences in population screened, recruitment process, and diagnostic criteria [2]. Although not a major problem for public health, reducing DH is related to improvement in oral health-related quality of life [3]. Brannstrom’s hydrodynamic theory remains the most widely accepted mechanistic explanation for the pathogenesis of DH [4, 5]. This theory posits that fluid movement induced by external stimuli stimulates nerve endings in the dental pulp, thereby resulting in tooth pain [6]. Given this etiology, DH is managed using two primary strategies: nerve desensitization, and dentin tubule occlusion [7, 8]. Potassium ions (K⁺), typically in the form of potassium nitrate, can depolarize nerve fiber membranes and render them less responsive to the stimuli-induced fluid movement [9]. Despite this established mechanism and its widespread use in dentifrices and mouth rinses, systematic reviews have indicated that strong clinical evidence supporting the superior efficacy of KNO₃ is still lacking compared to occlusion strategies [10, 11]. Furthermore, due to the need for sustained delivery for nerve depolarization, KNO₃ is rarely utilized in single-application professional agents like varnishes. Consequently, occlusion of the dentin tubules has emerged as a more robust and verifiable strategy. This approach directly addresses the hydrodynamic mechanism, using both chemical or physical methods to block the open orifices of the dentinal tubules, and reducing or eliminating the stimulus-induced fluid flow, thereby preventing the activation of the underlying nerves [11]. These occlusion strategies encompass both professional in-office treatments and daily home-care products. In clinical settings, professional agents such as low-viscosity resins are frequently utilized as a “gold standard” to achieve immediate and durable physical occlusion of dentinal tubules [12]. Conversely, for self-administered daily home care, a variety of active ingredients work through this mechanism, primarily focusing on biomimetic or mineralizing technologies, including hydroxyapatite (HAP), bioactive glasses (i.e., calcium sodium phosphosilicate), and arginine and calcium carbonate technology [11, 13].

Among the numerous occluding agents, HAP stands apart and has been increasingly utilized, primarily owing to its inherent biomimetic properties [14, 15]. As the principal inorganic mineral component of human enamel and dentin, it is considered one of the most biocompatible materials, being widely applied in dentistry for tooth remineralization [16–18]. The clinical effectiveness of HAP-based products, especially nano-sized, for managing DH is well-supported by a growing body of high-quality evidence [19–21]. A comprehensive systematic review and meta-analysis of 44 human clinical trials demonstrated that HAP significantly reduced dentin hypersensitivity by 39.5% compared to placebo, 23% compared to fluoride [13].

Strategies to enhance the desensitizing effect of HAP have traditionally focused on physical modifications like nano-sizing or chemical alterations like ion-substitution [22, 23]. However, recent advancements have introduced a novel, bio-inspired strategy using citrate as an active modulator of the mineralization process. Groundbreaking research in bone biology has identified citrate as a key organic molecule that is highly conserved in bone mineral (comprising 1.5-2.0 wt% of bone content) and is essential for normal bone formation [24, 25]. In vitro studies have demonstrated that citrate stabilizes prenucleation calcium phosphate clusters and liquid-like ACP precursors, preventing their premature conversion into crystalline HAP. This stabilization is vital, as it allows a more fluid, amorphous mineral phase to effectively infiltrate the intricate collagen fibril network of bone by reducing ACP-collagen interfacial energy before it solidifies into organized HAP crystals [26, 27]. Dayashankar et al. conducted a randomized controlled trial that compared a citric acid-based nanohydroxyapatite (CA-nHA) composite graft with a nanohydroxyapatite (nHA) graft alone for periodontal regeneration. The study found that while both materials showed significant improvement, the CA-nHA group demonstrated statistically significant better outcomes in all clinical and radiographic parameters at 12 months compared to the nHA-only group [25].

Building on the established role of citrate in bone biology, where amorphous calcium phosphate (ACP) precursors are stabilized to facilitate mineral infiltration, a similar mechanism in dentin remineralization is hypothesized. It is proposed that the efficacy of HAP-based dentinal tubule occlusion will be enhanced by citrate-modulated HAP formation. This process is expected to promote a deeper, more integrated mineral seal, rather than a superficial mineral accumulation limited to the tubule orifices. Such deep integration is critical for creating a durable protective layer on the dentin surface.

Therefore, this study aims to validate the superiority of the citrate-HAP complex over HAP alone through in vitro dentinal tubule occlusion assays, followed by a randomized clinical trial to assess its long-term efficacy against a placebo and a commercially available bioactive glass-based toothpaste, which will serve as the positive control. The null hypotheses tested were: (1) The citrate-HAP complex does not demonstrate significantly superior dentinal tubule occlusion compared to HAP alone in in vitro assays; and (2) There is no statistically significant difference in the reduction of dentin hypersensitivity between the citrate-HAP complex toothpaste and the control toothpastes (placebo and bioactive glass) in the clinical trial.

Materials and methods

In vitro dentinal tubule occluding

An in vitro study assessed dentinal tubule occlusion by toothpastes. Bovine incisors were used as a substrate for the in vitro dentin tubule occlusion model. The teeth were sourced from a commercial slaughterhouse in Bengbu, Anhui province, China. All teeth were collected from animals slaughtered for consumption and were considered by-products of the food industry. Only teeth with intact crowns and no visible cracks or caries were selected for the study. Using a low-speed precision saw (IsoMet^® 1000, Buehler, Lake Bluff, IL, USA) under water cooling, dentin blocks were sectioned from the cervical region, close to the pulp chamber. The obtained dentin discs measured approximately 5 mm in length and 1 mm in thickness. Thirty dentin discs were randomly divided into 3 groups through a simple randomization procedure: group A, test toothpaste (4.2% HAP, 5.53% potassium citrate monohydrate and 0.14% NaF), group B, in vitro control toothpaste (4.2% HAP and 0.14% NaF), and group C, placebo toothpaste (0.14% NaF only). To eliminate selection bias, the discs were pooled together and randomly picked by an investigator who was blinded to the specimen preparation process. The 3 formulas shared an identical base formulation (abrasives, humectants, etc.) with the only variables were HAP and citrate. To ensure baseline homogeneity between the test and control groups regarding tubule orientation and density, a split-specimen design was adopted. Each dentin disc was split longitudinally into two symmetrical halves. These halves were then etched with 40% phosphoric acid for 5 min to establish a hypersensitivity model with fully patent tubules. While similar in principle to the protocol described in Kulal’s report [28], our approach employed a more conservative etching duration to ensure the complete removal of the smear layer while avoiding excessive demineralization. For each pair, one half served as the control (immersed in artificial saliva), while the other half underwent the toothpaste treatment. This pairing ensured that the treated surface and its control originated from the same anatomical location.

The dentin discs were placed in a specially designed fixture used to hold an electric toothbrush (Oral-B Vitality D12; Procter & Gamble, Cincinnati, OH, USA) fitted with a sensitive toothbrush head (Model EB17S-4; Procter & Gamble), and a sensor for standardized brushing pressure was prepared. 0.5 g of toothpaste was applied to the dentin surface. The toothbrush bristles were positioned at a 90-degree angle to the specimen and maintained in contact with the dentin surface. Brushing pressure was set to 200 g. The dentin discs were brushed with toothpaste every morning and evening for 2 min each time. After the brushing procedure, the dentin discs were rinsed with deionized water for approximately 10 s to eliminate macroscopic paste residues, ensuring that only the occluding material within the tubules remained. The specimens were then kept in an artificial saliva (20 mM HEPES, 16 mM KCl, 1 mM CaCl₂·2H₂O, 4 mM KH₂PO₄, 4.5 mM NH₄Cl, 0.2 mM MgCl₂·6H₂O, pH = 7.0) incubator at 37 °C for the rest of the time.

Subsequent to the treatment, the specimens were dried and prepared for analysis by SEM. After drying, the discs were mounted onto aluminium stubs and subsequently coated with a thin layer of palladium in a sputter coater. The morphology of all specimens was examined under a scanning electron microscope of HITACHI S4800 at 10 kV acceleration voltage with magnifications between 1,000× and 10,000×. For the quantitative analysis of tubule occlusion, three random, non-overlapping fields of view were selected from the central region of each dentin disc by blindly moving the SEM stage to avoid edge effects and selection bias. Tubule occlusion percentage was calculated from these 1000x SEM images using the formula: [(n₀ - n₁)/n₀] X100%, where n₀ was the number of fully patent dentinal tubules in the untreated half dentin discs, and n₁ was the number of fully patent dentinal tubules in the treated half dentin discs. Statistical analysis was performed using a one-way analysis of variance (ANOVA), with a p-value of < 0.05 considered statistically significant.

Clinical study

Clinical process

This was a 12-week, double-blind, randomized, parallel design study. The study included three toothpaste groups: the test toothpaste (Group A: 4.2% HAP, 5.53% potassium citrate monohydrate, and 0.14% NaF), a placebo (Group C: 0.14% NaF only), and a positive control (Group D: a commercial bioglass-based anti-sensitivity toothpaste). Evaporative (air) stimulus (Schiff) and tactile stimulus (Yeaple) were used as evaluation indices. The study was conducted at the School & Hospital of Stomatology Wuhan University. The WUHAN University Institutional Review Board (IRB) approved the study ([2016] Ethics Review No. (01)). The trial was retrospectively registered on Chinese Clinical Trial Registry (No. ChiCTR2500115399, Date: 25/12/2025). The sample size was calculated based on the primary outcome (Schiff index) reported in previous literatures [20, 23]. The enrollment ratio was set to be 1:1:1, the alpha error (α) was ≤ 5%, and the test power (1-β) was 80%. Eligible participants were randomly assigned to one of the three study groups based on a random number table. Group assignments were concealed in sequentially numbered, opaque, sealed envelopes, which were opened only after the completion of baseline assessments to ensure allocation concealment. Male and female subjects aged 20–55 were accepted onto the study. Subjects must be absent from chronic diseases including but not limited to hypertension, diabetes mellitus, cardiovascular diseases, or autoimmune disorders, and are not taking any systemic medications that could interfere with pain perception or inflammatory response. Subjects must have symptoms of dentin hypersensitivity, with at least two teeth (in different quadrants and not adjacent) having exposed root dentin. Clinical assessments of dentine hypersensitivity were performed using standardized protocols [1, 11, 21]. Tactile threshold was measured using a calibrated electronic pressure-sensitive probe (Yeaple probe) applied to the sensitive area with forces increasing in 10-g increments until a pain response was elicited. Air-blast sensitivity was scored using the Schiff Scale (0–3) based on the subject’s response to a controlled cold air blast delivered from a distance of 1 cm for 1 s. Additionally, a 100-mm Visual Analog Scale (VAS) was used for subjects to self-report their subjective pain intensity immediately after stimulation. The left end (0 mm) represents “no pain” and the right end (100 mm) represents “worst possible pain.” When tested by Schiff cold air sensitivity scale, at least two teeth should have a score of 2 or higher, a VAS score of 4 or higher, and a sensitivity threshold of less than 20 g when tested with the Yeaple probe. Subjects meeting any of the following criteria will be excluded: severe oral diseases (Periodontitis, dental caries, oral mucosal diseases), chronic conditions, or allergies to the test product; progressive periodontal disease or periodontal treatment (including surgery) within the past year; sensitive tooth mobility greater than 1; extensive dental restorations, suspected pulpitis, dental caries, enamel crazing, or removable partial denture abutments; use of anticonvulsants, antihistamines, antidepressants, sedatives, tranquilizers, anti - inflammatory agents, or analgesics within the past month or currently; intermittent pain (e.g., back pain, arthritis); or participation in desensitizing toothpaste studies or regular use of other desensitizing toothpastes within the past three months. Informed consent from subjects was obtained.

There were totally of six examination visits to complete the study (Fig. 1). Subjects who met enrollment criteria would undergo a two-week washout period by using a standard fluoride toothpaste and designated soft-bristled toothbrush. They were assigned different test toothpastes after baseline examination, and continued to use it for 8 weeks and then changed to the washout standard fluoride toothpaste for another 4 weeks. The subjects were required to brush their teeth twice a day, filling the entire toothbrush head with toothpaste each time, and brushing their teeth for 2 min. At each examination visit, Yeaple and Schiff index were evaluated. For the Schiff test, a dental chair air gun (60 ± 5 p.s.i., 19–21 ℃) blows air for 1 second at 1 cm from the sensitive tooth (with a finger pressing the adjacent tooth to control variables), scored via the Schiff cold air sensitivity scale; for the Yeaple test, a calibrated electronic probe contacts exposed buccal dentin at the enamel-dentin junction, starting at 10 g and increasing by 10 g increments until discomfort (forces > 50 g are recorded as 60 g). One-way analysis of variance (ANOVA) was used to compare the differences between groups at different time points and within groups at the 5% significance level.

Fig. 1.

Overview of the clinical study protocol. The flowchart details the 12-week longitudinal study design, encompassing the initial screening and washout period, the 8-week test phase (Visits 2-5), and the subsequent 4-week follow-up evaluation (Visit 6) to assess long-term efficacy

Results

In vitro study

After treatment with 40% phosphoric acid solution for 5 min, all dentinal tubules were open. Cross-section images revealed smooth luminal surfaces within the tubules, with no obstructing particles observed (Fig. 2a and 2b). Following 3-day and 7-day treatments, dentin specimens immersed solely in artificial saliva showed no tubule occlusion (Fig. 3a and 3e). In contrast, all toothpaste-treated groups exhibited tubule occlusion (Fig. 3b, 3d, 3f-3h). After 3 days of toothpaste treatment, the dentinal tubule occlusion rates for Group A, Group B, and Group C were 96.0 ± 2.1%, 84.7 ± 4.8%, and 43.5 ± 10.1%, respectively. Significant differences in occlusion rates were observed among the three groups (p < 0.05). After 7 days of treatment, the occlusion rates increased to 99.7 ± 0.7% (Group A), 98.1 ± 1.7% (Group B), and 76.4 ± 7.3% (Group C), with statistically significant intergroup differences (p < 0.05).

Fig. 2.

SEM images of the dentin surface (a) and longitudinal section (b) after 5-minute treatment with 40% phosphoric acid

Fig. 3.

SEM images showing the top surfaces after 3 and 7 days treatment: (a) and (e) immersed only in artificial saliva for 3 and 7 days; (b) and (f) treated with group A for 3 and 7 days; (c) and (g) treated with group B for 3 and 7 days; (d) and (h) treated with group C for 3 and 7 days

Cross-section of the dentin showed that tubules immersed only in artificial saliva solution maintained smooth intratubular surfaces after both 3 and 7 days of treatment (Figs. 4a and 5a). After 3 and 7 days of treatment with test toothpaste (group A), the tubules exhibited intratubular occlusion depths of more than 30 μm and 40 μm, respectively. Additionally, mineralized deposit layers more than 3 μm and 5 μm thick formed on the surface (Figs. 4b and 5b). Figs. 4e and 5e are magnified views inside the dentinal tubules after treatment with group 1 for 3 days and 7 days, respectively. Mineralized particles can be seen within the dentinal tubules in both images. Occlusion depths of 10–15 μm were observed in the control group (Group B) at 3 and 7 days; however, a mineralized deposit layer of approximately 1 μm thickness formed on the surface only after 7 days (Figs. 4c and 5c). Only sparse particles were visible within the tubules of placebo treatment (group C), and no mineralized deposit layer formed on the surface at either time point (Figs. 4d and 5d). Compared to HAP control, citrate-HAP complex exhibited deeper mineral penetration into the dentinal tubules and a thicker mineral deposition layer on the dentin surface.

Fig. 4.

SEM images showing cross-sections of dentin discs after 3 days treatment: (a) immersed only in artificial saliva for 3 days; (b) and (e) treated with group A for 3 days; (c) treated with group B for 3 days; (d) treated with group C for 3 days

Fig. 5.

SEM images showing cross-sections of dentin discs after 7 days treatment: (a) immersed only in artificial saliva for 7 days; (b) and (e) treated with group A for 7 days; (c) treated with group B for 7 days; (d) treated with group C for 7 days

Clinical study

One hundred twenty nine subjects from Wuhan city completed the trial and no adverse reactions were reported. The demographic information about the study population was shown in Table 1. The statistical significance of the clinical outcomes was verified by both parametric and non-parametric tests, which produced consistent conclusions. For clarity and ease of data visualization, the results are presented based on the one-way ANOVA analysis. Both the novel HAP/Citrate toothpaste (group A) and the positive control (group D) showed significant DH reductions in average Schiff and Yeaple indices vs. baseline after 6 and 8 weeks (p < 0.05). After 6 weeks of use, the Schiff index of group A and group D was significantly improved compared with the placebo control group (p < 0.05), the improvement was 21.39% and 28.44% for 6 weeks, respectively. After 8 weeks of use, the Yeaple index of the test toothpaste group and the positive control group was significantly improved compared with the placebo control group (p < 0.05), the improvement was 100.85% and 69.17%, respectively. By 8 weeks, Schiff index improvements remained statistically significant (p < 0.05), and even 4 weeks after switching to regular fluoride toothpaste (12-week mark), both groups maintained Schiff index differences from baseline (p < 0.05). For the Yeaple index, while both groups showed significant 8-week changes versus the negative control (p < 0.05), these improvements decreased after switching to regular toothpaste. Group A showed larger average Yeaple index reductions by the 8-week mark, indicating superior late-stage efficacy for tactile stimulus sensitivity. Detailed data can be found in Tables 2 and 3.

Table 1.

Age and gender characteristics of the study population

Product	Number of subjects			Age
	Male	Female	Total	Mean	Scope
A	7	35	42	42	21 ~ 54
C	5	39	44	42	29 ~ 54
D	6	37	43	41	25 ~ 55

Table 2.

Clinical study results: mean (s.d.) Schiff and Yeaple index values at each test session

Index	Product	Baseline	2 weeks	6 weeks	8 weeks	12 weeks
Schiff	A	2.25(0.37)	2.13(0.39)	1.75(0.53)^*	1.51(0.67)^*	1.18(0.73)^*
	C	2.32(0.57)	2.26(0.39)	2.23(0.42)	2.15(0.46)	2.09(0.5)^
	D	2.29(0.43)	2.13(0.46)	1.59(0.63)^*	1.38(0.67)^*	1.07(0.70)^*
Yeaple	A	11.48(2.77)	10.91(2.23)	15.91(8.16)^	28.69(13.43)^*	23.75(15.29)^
	C	11.43(2.54)	11.07(2.35)	12.62(6.92)	14.29(6.95)	20.73(13.26)^
	D	11.28(2.46)	11.43(3.71)	15.93(9.4)^	24.17(13.57)^*	20.70(13.52)^

^{^}means the mean values are significantly different with baseline (p < 0.05); ^*means the mean values are significantly different with control group C (p < 0.05)

Table 3.

The improvement rate^ of Schiff and Yeaple for group A and group D compared with control group C

Index	Group	2 weeks	6 weeks	8 weeks	12 weeks
Schiff	A	6.05%	21.39%	29.86%	43.34%
	D	5.79%	28.44%	35.91%	48.71%
Yeaple	A	-1.47%	26.07%	100.85%	14.56%
	D	3.23%	26.24%	69.17%	-0.16%

^{^}Improvement rate= (Group A/D-Group C)/ Group C ×100%

Discussion

This study demonstrates how the novel hydroxyapatite-citrate complex works better than HAP alone in treating dentin hypersensitivity. In vitro study shows that the HAP-citrate complex significantly enhances dentinal tubule occlusion. It leads to a higher tubule occlusion rate, substantially deeper intratubular mineral penetration and the formation of a thicker mineralized surface layer.

The lab findings provide direct support for the hypothesis that citrate acts as an active modulator of mineralization. Firstly, the multi-carboxylic structure of the citrate ions acts as smart anchors to chelate HAP particles [29], which may help HAP to bind more effectively to the dentine surface and tubule inner surface for targeted deposition. Thereafter, HAP triggers an active remineralization process, providing both crystal nuclei and a sustained supply of calcium and phosphate ions [15]. At this stage, citrate also works to stabilize the ACP precursors [23, 30], allowing a more fluid mineral phase to penetrate deeper into the tubules before solidifying.

This formulation offers a dual-action approach to managing hypersensitivity. The K⁺ from potassium citrate provides the effect of nerve desensitization, offering symptomatic relief by blocking pain signal transmission. At the same time, the HAP-citrate complex tackles the root cause of DH by building a long-lasting tubule occlusion and enhancing the biomimetic remineralization process. This combination of powerful in vitro occlusion and the dual therapeutic action explains the positive clinical results we observed.

In the clinical trial, the HAP-citrate toothpaste delivered significant relief from both air (Schiff) and tactile (Yeaple) stimuli compared to the placebo, with improvements seen as early as 6 weeks. The desensitizing effect of HAP-citrate toothpaste was comparable to the positive control, a commercial bioactive glass toothpaste, showing its strong clinical efficacy. There was an important finding during this clinical process: the reduction in air-stimulus sensitivity not only held steady across the 8-week study duration but, remarkably, persisted even after a 4-week period using a placebo fluoride toothpaste. However, while the desensitizing effect against air stimuli (Schiff index) remained stable at week 12, the tactile threshold (Yeaple index) showed a decline after the 4-week washout period. We hypothesize that this divergence results from the different physical locations of the mineralized deposits. The deep intratubular plugs (> 40 μm) observed in our SEM analysis are likely well-protected from oral clearance, thus maintaining a long-term reduction in hydrodynamic fluid flow. Conversely, relief from tactile stimuli may be more dependent on the coverage of the superficial dentin surface. During the washout period, the lack of active HAP-citrate replenishment may have led to the gradual mechanical removal of this superficial mineralized layer by daily toothbrushing. This speculation, while consistent with our clinical and SEM findings, requires further investigation using dynamic wear-rate models to confirm the longitudinal stability of surface versus deep mineralization. It is important to note that the 12-week Yeaple score (23.75 ± 15.29) remains significantly superior to both the baseline (11.48 ± 2.77) and the 6-week results (15.91 ± 8.16), confirming a “persistent but attenuating” protective effect. The high SD suggests that the duration and intensity of the “persistence effect” vary significantly between individuals. This variation is likely due to differences in individual oral conditions, such as the force applied during toothbrushing (which affects the rate of surface layer wear), salivary flow rate and composition (which influence mineral dissolution), and dietary habits.

Although these results are highly encouraging, this study has limitations that must be acknowledged. First, concerning the clinical design, a primary limitation is the lack of a HAP-only toothpaste control group. While the in vitro results strongly support the synergistic mechanism of citrate and HAP, the clinical “synergy” is inferred from the superior performance of the complex compared to literature values, rather than a direct side-by-side clinical comparison. Additionally, our clinical evaluation relied on objective (Schiff) and subjective (Yeaple) sensitivity scores but did not include an assessment of Oral Health–Related Quality of Life (OHRQoL), which is considered a key outcome parameter for understanding the broader impact of DH treatment on patient well-being [31].

Second, regarding the in vitro methodology, the experimental design focused exclusively on comparing strictly matched base formulations (HAP with and without citrate) to scientifically isolate the synergistic mechanism. Consequently, the study did not include a commercial positive control (such as the bioglass-based toothpaste used in the clinical phase) for direct comparison, which limits the comparative interpretation of the SEM results against established commercial occluding agents. In addition, the stability of the mineral plugs was not explicitly tested under acid challenges or long-term mechanical brushing conditions. It remains to be investigated how stable these plugs are under changing pH conditions mimicking the oral environment. Regarding the substrate, bovine incisors were used as a model. While bovine dentin is a widely accepted substitute in dental research, it exhibits differences in tubule density compared to human dentin, although tubule diameters are generally considered comparable [32]. Furthermore, for the observed plugs, we relied on morphological evidence and did not perform energy-dispersive X-ray (EDX) analyses to verify the elemental composition (HAP vs. smear plugs) [33]. Future studies should aim to address these gaps. Specifically, hydraulic conductance measurements (e.g., Flodec) [34] could be employed to quantify the reduction in fluid flow, thereby providing direct verification of the hydrodynamic mechanism and further validating the superior anti-sensitivity efficacy of the HAP-citrate complex.

This study combines in vitro and clinical research methods, presenting a promising new formulation for the treatment of dentin hypersensitivity. The hydroxyapatite-citrate toothpaste exhibits excellent tubule occlusion efficacy in vitro and effectively provides long-lasting relief from dentin hypersensitivity in the clinical trial. We speculate that citrate stabilizes the calcium phosphate clusters and liquid ACP precursors before nucleation, preventing them from prematurely converting into crystalline HAP, leading to the formation of a highly integrated and robust dentinal seal. It is this biomimetic, depth-percolating mechanism that accounts for the remarkable persistence of the clinical desensitizing effect, especially the sustained reduction of air-stimulus sensitivity observed in the four weeks placebo toothpaste period.

Conclusions

These studies demonstrate that the HAP-citrate complex is a highly effective biomimetic formulation for managing dentin hypersensitivity. Our in vitro results provide direct evidence of a synergistic mechanism, where citrate acts as a modulator to significantly enhance HAP-mediated tubule occlusion, forming a dense mineralized seal reaching depths of over 40 μm. Clinically, this formulation translates these mechanistic advantages into significant therapeutic performance, delivering rapid relief from both tactile and evaporative stimuli and maintaining a sustained desensitizing effect even after product discontinuation. While the specific clinical increment of the synergy (compared to HAP alone) remains to be fully elucidated through future investigation, the combined findings validate the HAP-citrate complex as a science-backed and durable treatment strategy.

Abbreviations

HAP

hydroxyapatite

DH

dentine hypersensitivity

Authors’ contributions

Xiaobin Chen and Han Jiang contributed equally to this work and are considered co-first authors. Xiaobin Chen and Minquan Du conceived and designed the study. HJ was responsible for the execution of the clinical trial and data collection. Yanxiao Li performed the in vitro experiments and SEM analysis. Yi Zhou and Decheng Ye participated in statistical analysis and clinical coordination. Xiaobin Chen drafted the manuscript, and Minquan Du provided revisions for important intellectual content. All authors read and approved the final manuscript.

Funding

This study was funded by Hawley & Hazel Chemical Co. (Zhongshan) Ltd.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

For the in vitro portion of this study, bovine teeth were obtained from a commercial slaughterhouse. No animals were sacrificed specifically for this research. As the tissues were by-products of the food industry, formal ethical approval for animal use was not required. The clinical study was conducted at the School & Hospital of Stomatology Wuhan University and was approved by WUHAN University Institutional Review Board (IRB) ([2016] Ethics Review No. (01)).

Consent for publication

All involved subjects agreed to the publication.

Competing interests

Xiaobin Chen, Yanxiao Li, Yi Zhou, Decheng Ye are employees of Hawley & Hazel Chemical Co. (Zhongshan) Ltd. Han Jiang and Minquan Du declare that they have no competing interests.