Introduction
Dentin hypersensitivity (DH) is a prevalent and widely concerning problem in dentistry. This condition causes significant discomfort for patients and can severely affect quality of life, with a reported prevalence rate as high as 57% among adults.1 DH arises due to exposed dentinal tubules (DTs), and various external factors may exacerbate the symptoms, such as daily exposure to temperature changes, chemical agents in the oral environment, and mechanical forces from chewing and toothbrushing.2 Current clinical treatments primarily include pharmacological therapy, laser therapy, restorative approaches, and surgical interventions such as root canal treatment (RCT) for refractory cases.3,4 However, these methods often demonstrate insufficient efficacy, limited duration of effect, or narrow applicability, which highlights the need for more effective treatment strategies in dental research.
Approaches to occlude DTs for the management of DH generally include surface ion precipitation to form a sealing layer, biomimetic remineralization to generate hydroxyapatite (HAP) crystals within the tubules, and protein precipitation for tubule occlusion. Among these, biomimetic remineralization has attracted significant attention from researchers due to several advantages.5,6 This approach closely mimics the natural physiological mineralization process,7,8 and precise control over mineralization conditions allows the mineralized layer to exhibit excellent mechanical properties and stability, ensuring durable isolation from external stimuli, which is essential for alleviating DH symptoms.9,10 Biomimetic approaches, such as microwave-synthesized chicken eggshell-derived nano-hydroxyapatite (CEnHAp) combined with phytosphingosine (PHS) or eggshell-derived nanohydroxyapatite (EnHA) combined with carboxymethyl chitosan (CMC), enable superior remineralization.11,12 These strategies replicate natural mineralization processes, forming stable, high-performance mineralized layers that effectively block external stimuli, and offer promising therapeutic solutions for DH.
Dentin mineralization is classified into intrafibrillar and interfibrillar mineralization.13 Intrafibrillar mineralization primarily refers to the deposition of minerals within the interstitial regions of collagen fibers and their orderly arrangement parallel to the c-axis of the fibers. In contrast, interfibrillar mineralization is characterized by the disordered deposition of minerals between collagen fibers.14 Intrafibrillar mineralization is fundamental to dentin biomineralization and is generally more favorable.15,16 Dentin sialophosphoprotein (DSPP), a critical non-collagenous protein (NCP), orchestrates dentin mineralization through its highly anionic domains. DSPP contains two functional domains: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). The phosphate and carboxyl groups in DPP specifically facilitate calcium binding and nucleation, whereas the protein’s collagen-binding capacity directs intrafibrillar mineral deposition.17,18 In addition to DSPP, amorphous calcium phosphate (ACP), collagen fibers, and other NCPs are essential during intrafibrillar mineralization.19,20
Polyelectrolytes, as NCP analogs, share similar structural and physicochemical properties,21,22 enabling stabilization of nanoscale calcium phosphate in an amorphous phase and facilitating biomimetic mineralization.23,24 For instance, the anionic polyaspartic acid (Pasp) mimics DSPP by trapping calcium ions (Ca²⁺) via its amino (-NH₂) groups, forming stable ACP and promoting intrafibrillar mineralization.25–27 Recent studies highlight the critical role of nanoscale calcium phosphate in enhancing intrafibrillar mineralization. Calcium phosphate particles stabilized by polyacrylic acid (PAA) and Pasp have achieved 95% collagen mineralization in vitro and demonstrated substantial regenerative effects in osteoporotic bone defects.28
Dentin, in contrast to enamel, contains a lower mineral content and is more vulnerable to cariogenic bacteria.29 These bacteria and their metabolic byproducts can rapidly penetrate dentin and reach the root canals through exposed DTs, leading to infectious diseases in dentin, periodontium, pulp, and periapical regions.30,31 The adhesion of cariogenic bacteria on the dentin surface also initiates continuous mineral dissolution and loss.32,33 Most treatment strategies for DH focus on pain control, and insufficient attention is often paid to the bacterial challenges in exposed dentin. Effective bacterial control in the exposed dentin area should not be overlooked for patients with DH.
CMC is an amphoteric polyelectrolyte containing carboxyl (-COOH) and amino (-NH2) functional groups, functioning as either an anionic or cationic polyelectrolyte depending on whether the pH is below or above its isoelectric point (PI = 3.5).34,35 CMC has been shown to stabilize ACP at pH 7 and significantly promote the intrafibrillar mineralization of collagen fibers.14,36 In addition, CMC possesses antimicrobial activity. It can inhibit the growth of various bacteria, such as Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa), potentially by disrupting bacterial cell walls and membranes or inhibiting metabolic processes.37 CMC can also form a uniform and stable film, providing favorable mechanical and barrier properties.38
In summary, this study aims to utilize Pasp and CMC to compete for Ca²⁺, effectively inhibiting the rapid precipitation of Ca²⁺ and phosphate ions (PO₄³−) and thereby forming highly stable nano-ACP. This mechanism not only synergistically regulates the directional growth and morphology of mineral crystals but also suppresses the proliferation of cariogenic bacteria through the antibacterial properties of CMC. By integrating the dual effects of DT occlusion and antibacterial action, this multifunctional composite is expected to achieve superior tubule sealing and antibacterial efficacy compared to traditional desensitizing agents. This strategy offers a new approach for the synergistic prevention and treatment of DH and dental caries.
Materials and Methods
Pasp (Rhawn, China), CMC (degree of substitution 0.8, degree of deacetylation 85%, molecular weight 100 kDa, Aladdin, USA), calcium chloride solution (CaCl2, 2.5 mol/L, Macklin, China), dibasic sodium phosphate solution (Na₂HPO₄, 1.5 mol/L, Leagene, China), ethanol (analytical reagent grade, Sinopharm Chemical Reagent Co., Ltd., China), Tris-buffered saline (TBS, Solarbio, China), type I collagen lyophilized powder (rat tail, MedChemExpress, USA), acetic acid (analytical reagent, Sinopharm Chemical Reagent Co., Ltd., China), sodium hydroxide (NaOH, Aladdin, China), HEPES buffer (Biorigin, China), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, Macklin, China), thymol (analytical reagent, Rhawn, China), ethylenediaminetetraacetic acid (EDTA, Solarbio, China), Gluma desensitizer (Gluma, Germany), artificial saliva (Leagene, China), citric acid (Codow, China), Brain Heart Infusion broth (BHI, Hopebio, China), agar powder (Solarbio, China), bacterial viability assay kit with DMAO and propidium iodide (PI) (Beyotime, China), phosphate-buffered saline (PBS, Pricella, China), fetal bovine serum (FBS, Pricella, China), penicillin-streptomycin solution (Pricella, China), α-MEM medium (Pricella, China), Cell Counting Kit-8 (MedChemExpress, USA), and Calcein AM/PI Double Staining Kit (Elabscience, China) were used in all experiments.
Preparation of Polyelectrolyte Complexes
At room temperature, 0.4 g/mL Pasp and 0.15 g/mL CMC solutions were first prepared. Next, 1.6 mL of CMC solution was combined with 0.27 mL of Pasp solution, followed by the addition of 0.5 mL CaCl2 solution, and stirred thoroughly to obtain Solution I. Separately, 1.6 mL of CMC solution was mixed with 0.5 mL Na2HPO₄ solution and stirred to obtain Solution II. The PCA composite was formed by mixing Solutions I and II with constant stirring. Control complexes were synthesized using the same method: PA without CMC, CA without Pasp, and PC without both CaCl₂ and Na₂HPO₄. The pH of all solutions was adjusted to neutral using 0.1 M NaOH or HCl.
Characterization of Polyelectrolyte Complexes
PCA, PA, CA, and PC samples were frozen at –80°C for 24 h and lyophilized using a freeze dryer (Alpha 2–4LSC BASIC, Germany) for another 24 h. The freeze-dried samples were dispersed ultrasonically in ethanol (20 kHz, 2 min), dropped onto transmission electron microscopy (TEM) copper grids, and observed using high-resolution TEM (HRTEM, JEM 2100F, Japan) at 100 kV. The diameter distribution of calcium phosphate nanoclusters was analyzed with ImageJ software. Crystalline phases were identified by selected-area electron diffraction (SAED, JEM 2100F, Japan; camera length 100 cm, exposure 2 s). Surface morphology was examined by scanning electron microscopy (SEM, Tescan Vega3, Czech Republic) at 7 kV and 8 mm working distance. Elemental composition and distribution were evaluated by energy-dispersive spectroscopy (EDS, Oxford Instruments X-Max 50, Czech Republic) at 15 kV, 10 mm working distance, and 60s acquisition. X-ray diffraction (XRD, Ultima IV, Japan) was conducted over a 2θ range of 10–70° at 10°/min. Zeta potential was measured using a Zetasizer Nano ZS (UK) at 25°C and 13° scattering angle. Fourier transform infrared spectroscopy (FTIR, Nicolet iS50, USA) was used to characterize functional groups through 32 scans (wavenumber range: 400–4000 cm−¹, resolution: 4 cm−¹).
In vitro Release of Calcium and Phosphorus
To minimize interference from artificial saliva, simulated body fluid (SBF), and PBS, 1 mL of PCA, PA, and CA were each immersed in 10 mL TBS. To simulate physiological body temperature, the mixtures were stirred at 37°C for 10 min, then incubated under static conditions in TBS for 24 h. After incubation, samples were centrifuged at 4000 rpm for 10 min, and 5 mL supernatant was collected from each sample. The supernatants were subjected to microwave-assisted digestion prior to analysis. Quantitative determination of released calcium (Ca) and phosphorus (P) was performed using inductively coupled plasma optical emission spectrometry (ICP-OES, Agilent 5800).
Intrafibrillar Mineralization of Reconstituted Collagen
Type I collagen (1 mg/mL) was prepared by dissolving collagen lyophilized powder in 0.1 M acetic acid at 4°C. Two hundred-mesh nickel TEM grids were immersed in the collagen solution, and pH was adjusted to neutral with 1 M NaOH. HEPES buffer (pH 7.4) was added to promote collagen self-assembly, followed by immersion in 0.3 M EDC solution for 4 h to induce crosslinking. Grids were rinsed with ultrapure water and air-dried. For remineralization, 1 mL of PCA, PA, CA, and PC was each added to 10 mL ultrapure water and shaken for 30 min to release ACP. The nickel grids were immersed in each remineralization solution and incubated at 37°C for 1, 3, or 5 days. After mineralization, the grids were washed, negatively stained with uranyl acetate, and analyzed using TEM (JEM-1400Flash, Japan) to assess collagen mineralization and by SAED to determine the crystallinity of CaP minerals.
Preparation of DH Models
A power analysis was performed using G*Power 3.1 with an effect size of 0.4 and a significance level of α = 0.05. The analysis determined that a minimum sample size of 90 would be necessary to achieve 80% statistical power for detecting significant effects. To enhance statistical power and accommodate potential attrition, the final sample size was increased to 120.
The protocol was approved by the Medical Ethics Committee of the Affiliated Hospital of Qingdao University (QYFYWZLL29632). After obtaining informed consent, 120 caries-free, crack-free third molars freshly extracted for orthodontic or occlusal reasons were collected within 1 month. Teeth were preserved in thymol solution at 4°C after removing root surface soft tissue and tartar with a curette. Dentin discs (5 × 5×1 mm) were prepared from the third molar along the long axis using a low-speed precision cutting machine (DTQ-5, China), with the distal pulpal surface as the experimental surface. Discs were sequentially polished with 600, 800, 1200, 2000, and 3000 grit silicon carbide sandpaper and rinsed in ultrapure water to achieve a smooth surface. Each disc was treated with 0.5 M EDTA (pH 8) for 5 min and ultrasonically cleaned with ultrapure water for 10 min.
Grouping of Demineralized Dentin Discs
Dentin discs were prepared following standardized protocols and sequentially numbered. To ensure unbiased allocation, a computer-generated randomization process was conducted under independent supervision. An investigator who did not participate in subsequent procedures generated the randomization schedule using a random number table, ensuring equal probability of assignment for each disc. Therefore, all dentin discs were randomly assigned to groups of 20 samples each: A (blank), B (Gluma desensitizer), C (PCA), D (PA), E (CA), and F (PC).
For Group A, the surface of each dentin disc was brushed for 60s, left to air dry, and rinsed with pressure-free ultrapure water. Group B received Gluma desensitizer applied for 60s with a small brush; the surface was rinsed with pressure-free ultrapure water after the liquid film disappeared and the surface lost its shine. Groups C, D, E, and F were treated using the same protocol with PCA, PA, CA, and PC. All groups received daily treatment for 28 days and were stored in fresh artificial saliva at 37°C after each application.
SEM Observation of DT Occlusion
At 28 days of treatment, dentin discs were randomly selected from each group. After rinsing with ultrapure water, samples were dehydrated through a graded ethanol series (30%, 50%, 70%, 80%, 90%, 95%, and 100%). Drying was performed in a forced convection oven (DHG-9000, China) at 60°C. Gold sputter-coating was applied prior to SEM analysis to assess DT occlusion, and EDS was used to determine the calcium and phosphorus contents. After 28 days, additional dentin discs from each group were selected. To avoid affecting the experimental surface, each disc was sectioned from the untreated side to a depth of approximately 0.2 mm and split by external force. After further rinsing, dehydration, drying, and gold coating, SEM was used to observe tubule occlusion in cross-section. The resulting bisected sections were allocated to either acid immersion or abrasion resistance testing. Acid resistance was evaluated by immersing samples in 6% citric acid solution (pH 1.5) for 1 min. Abrasion resistance was assessed by simulated toothbrushing with a soft-bristled brush under controlled force (2 min per session, twice daily for 7 days). Treated discs were then rinsed, dehydrated, dried, gold coated, and examined by SEM.
The Examination of Dentin Airtightness
A modified airtightness device was employed to evaluate the macroscopic effect of tubule occlusion. Each dentin disc was clamped between two rubber rings with a central opening and fixed in the chamber. A droplet of water was placed in a tube connected to the chamber. Air was injected using a syringe, and a manometer recorded the air pressure at which water movement occurred, defined as the critical pressure. Higher pressure indicated lower dentin permeability and more effective occlusion. Three samples per group were tested, with five measurements for each sample. The airtightness test was repeated after acid and abrasion testing.
The Examination of Dentin Composition Hardness
Native dentin, demineralized dentin, and dentin discs from Groups A–F were tested for hardness using the Vickers hardness test (Wilson Tukon1102, USA). A diamond indenter (apex angle 136°) applied a load of 0.5 kgf for 10s. The length of the two diagonals of each indentation was measured microscopically. Three samples per group were tested, with three measurement points for each sample. The Vickers hardness value (HV) was calculated using the mean diagonal length and a standard reference table.
Preparation of Dental Plaque Biofilm
Streptococcus mutans (S. mutans, ATCC25175 = NCTC10449) was obtained from the Shanghai Biology Collection Center (SHBCC) for biofilm formation. The suspension of S. mutans was cultured anaerobically in BHI medium at 37°C with 5% CO2. The optimal dilution (107 CFU/mL) was established, and 1% sucrose was added to the inoculation medium. Dentin discs treated for 28 days in Groups A, C, D, E, and F were sterilized by UV irradiation (254 nm) for 72 h, placed in 24-well plates with 1 mL inoculation medium, and incubated anaerobically at 37°C for 36 h to form biofilm. Discs were rinsed three times with PBS to remove non-adherent bacteria, transferred to new 24-well plates, and labeled accordingly.
Anti-Bacterial Activity
To the corresponding groups (A, C, D, E, F), 100 μL of ultrapure water, PCA, CA, or PC was added to their respective media containing biofilm-inoculated dentin discs. Plates were incubated anaerobically for 24 h. Following incubation, discs were rinsed three times with PBS, and the biofilms were stained with a bacterial viability assay kit (DMAO & PI) according to manufacturer instructions. Confocal laser scanning microscopy (CLSM, Olympus FV1200, Japan) was used to visualize live and dead bacteria, and quantitative analysis was performed with ImageJ.
To further assess the antimicrobial effect, incubated discs were fixed in 2.5% glutaraldehyde, rinsed, and dehydrated through graded ethanol. After drying and gold sputter-coating, samples were imaged by SEM. Three representative images per group were analyzed to evaluate bacterial adhesion.
For quantitative bacterial counts, biofilm was dislodged from each disc by vortexing in 3 mL PBS for 5 min. For further assessment, 0.1 mL of PCA, CA, and PC was co-cultured with 1 mL S. mutans suspension at 37°C for 12 and 24 h. Each suspension and the control were serially diluted in PBS (10−1 to 10−6), and 100 μL was plated on BHI agar. Plates were incubated anaerobically at 37°C for 48 h, and colonies were counted. Each assay was performed in triplicate.
Cultivation of Human Dental Pulp Stem Cells
This study was conducted in accordance with ethical approval requirements (QYFYWZLL29632). After obtaining informed consent, 30 caries-free, crack-free premolars were collected from healthy adults aged 18–25 years, extracted for orthodontic or impaction reasons. Under sterile conditions, dental pulp tissue was isolated, and human dental pulp stem cells (HDPSCs) were harvested using the tissue explant method. Primary cultures were maintained in α-MEM complete medium supplemented with 10% FBS. Third-passage cells were analyzed for surface markers (CD73, CD90, CD105, CD34, CD45) by flow cytometry (CytoFLEX, China) to confirm compliance with international standards for dental pulp stem cell identification.
Cytotoxicity Test
Extracts of PCA, PA, CA, and PC were prepared at a 1:10 (v/v) ratio in culture medium. The mixtures were incubated at 37°C, 100 rpm for 72 h to facilitate extraction of leachable components. After incubation, the extracts were filtered through a 0.22 μm sterile membrane to remove particulates, yielding final extracts for further use.
Third-passage HDPSCs (1 × 105 cells) were resuspended in 2.5 mL medium and seeded into 35 mm glass-bottom dishes. After 24 h of incubation at 37°C in a humidified 5% CO2 environment, the medium was replaced with PCA, PA, CA, or PC extracts (1:10, v/v), and cells were cultured for an additional 48 h. Following treatment, the medium was removed, and the cells were washed twice with PBS. Calcein AM/PI staining working solution (400 μL) was added, followed by 30 min of incubation in the dark. The staining results were observed under CLSM, and live/dead cell counts were quantified using ImageJ.
In parallel, third-passage HDPSCs were seeded into 96-well plates (5 × 10³ cells/well) and incubated for 24 h. Cells were then treated with PCA, PA, CA, or PC extracts (1:10, v/v) for 1, 3, and 5 days. At each time point, the medium was replaced with fresh medium containing 10% Cell Counting Kit-8 (CCK-8) reagent and incubated for 2 h. Optical density (OD) was measured at 450 nm, and results were normalized to the control group.
Hemolysis Test
This study was conducted in accordance with ethical approval (QYFYWZLL29632). Healthy human blood samples were collected from the Laboratory Department. One milliliter of blood was diluted with 7 mL PBS, gently mixed, and centrifuged at 1500 rpm for 5 min. The supernatant was removed, and PBS was added to the 8 mL mark along the tube wall. This washing process was repeated four times. On the final wash, no PBS was added after removing the supernatant, resulting in a suspension of red blood cells. Sample preparation included: (A) pure red blood cells and (B) a red blood cell/PBS solution (10%). Test groups were as follows: (i) positive control (total hemolysis), 50 µL A with 950 µL distilled water; (ii) negative control, 500 µL B with 500 µL PBS; (iii) polyelectrolyte complex groups, 500 µL B with 500 µL of each of the PCA, PA, CA, or PC solutions that had been immersed in PBS for 24 h. Each group and control was prepared in triplicate. All samples were incubated at 37 °C for 1.5 h, then centrifuged at 3000 rpm for 15 min. Supernatants were transferred to a 96-well plate, and absorbance was measured at 540 nm. The hemolysis rate (%) was calculated as follows:
Hemolysis rate = (OD polyelectrolyte complex – OD negative) / (OD positive − OD negative) × 100%.
Hemolysis rates ≤ 5% meet safety requirements for clinical use.
Statistical Analysis
All data are presented as mean ± standard deviation from at least three independent experiments. Normality was assessed by the Shapiro–Wilk test, and homogeneity of variance was verified with Levene’s test. For data meeting normality (p ≥ 0.05) and homogeneity, one-way ANOVA with Tukey’s post hoc test was applied. For non-parametric data, the Kruskal–Wallis test with Dunn’s post hoc correction was used. A two-sided p < 0.05 was considered statistically significant (α = 0.05). Significance levels are indicated as ns (not significant), p < 0.05, or p < 0.01. All analyses were performed using IBM SPSS Statistics 27.
Results and Discussion
This study investigated the synergistic mechanism of Pasp and CMC in dentin biomimetic mineralization by constructing their composite system. Experimental results demonstrated that these two polyelectrolytes competitively bind Ca²⁺ ions to form polyelectrolyte complexes (PCA) containing nanoscale ACP. As biomimetic analogs of NCPs, Pasp and CMC effectively stabilized ACP prenucleation clusters and regulated their structure and morphology, enhancing mineralization activity.14,39 Notably, the incorporation of CMC not only participated in the mineralization process but also inhibited dental plaque biofilm formation through its antibacterial properties. This characteristic is crucial for promoting the penetration of mineralization precursors into dentin.40 The supplemented Ca²⁺ and PO₄³− ions in the composite system compensated for the deficiency of mineralizing ions in saliva, enhancing overall mineralization capacity.41,42 In material design, this study systematically compared the performance differences between PCA and three other complexes (PA, CA, and PC) to thoroughly evaluate their application potential in treating DH and caries prevention.
Characterization using HRTEM and SAED revealed the presence of amorphous material in PCA, PA, and CA samples (Figure 1A1–A4), whereas no such component was detected in the PC group (Figure S1A). HRTEM observation showed that the amorphous phase in PCA exhibited the smallest particle size, approximately 2 nm in diameter, which was significantly smaller than those in the PA and CA groups (Figure 1A2). Further SEM coupled with EDS analysis confirmed this amorphous phase as ACP (Figure 1B1–B3, C1–C3, D1–D3), with no ACP characteristic signals observed in the PC group (Figure S1B). The nanoscale ACP clusters in PCA may facilitate better penetration into DTs, suggesting superior tubule-occluding potential. However, the gel-like properties of PCA may partially limit its penetrability, which requires further experimental verification. Of particular note were the Ca/P ratios: PCA (1.67) closely matched the theoretical value for HAP (1.67), whereas PA (1.93) and CA (2.54) showed significant deviations (Figure S2). Given that HAP is the primary inorganic component of dentin, the Ca/P ratio of PCA suggests its potential advantage in promoting biomimetic mineralization. This finding provides critical evidence for PCA’s remineralization performance.
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Figure 1 Characterization of PCA, PA, CA, and PC. (A1) TEM image of PCA (inset shows SAED pattern). (A2) Local magnified TEM image of A1. TEM images of (A3) PA and (A4) CA (insets show SAED patterns). (B1) SEM image of PCA. EDS mapping images of PCA, (B2) calcium, and (B3) phosphorus. (C1) SEM image of PA. EDS mapping images of PA, (C2) calcium, and (C3) phosphorus. (D1) SEM image of CA. EDS mapping images of CA, (D2) calcium, and (D3) phosphorus. (E) XRD image. (F) Zeta potential image. (G) Release of Ca and P ions. Different letters denote significant differences (p < 0.05). (H1-3) FTIR spectra. (See Figure S1 for TEM and SEM images of PC and Figure S3 for FTIR spectra of PC).
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Figure 2 Influence of PCA, PA, CA, and PC on reconstituted collagen. When incubated in PCA for (A1) 1, (A2) 3, and (A3) 5 days, collagen fibers exhibited slight mineralization at 1 day. At 3 days, most collagen fibers were mineralized; at 5 days, the collagen fibers were mineralized entirely. The SAED presented an arcuate diffraction pattern in the (002) and (211) planes. After being incubated in PA for (B1) 1, (B2) 3, and (B3) 5 days, the degree and rate of collagen fiber mineralization were lower than those in PCA. Nevertheless, at 5 days, SAED showed arc-shaped diffraction patterns in the (002) and (211) planes, though these patterns were less distinct than those in PCA. Incubation in CA for (C1) 1, (C2) 3, and (C3) 5 days demonstrated the weakest degree and rate of collagen fiber mineralization. Only some fibers were mineralized at 5 days. During incubation in PC for (D1) 1, (D2) 3, and (D3) 5 days, collagen fibers were not mineralized.
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XRD analysis confirmed the amorphous nature of PCA, PA, and CA (Figure 1E). Specifically, broad characteristic peaks were observed at 2θ = 25° for PCA, 2θ = 30° for PA, and 2θ = 24° for CA, consistent with the diffraction pattern of ACP. No distinct crystalline diffraction peaks corresponding to HAP or other crystalline calcium phosphate phases were detected, further confirming the amorphous characteristics of these materials.
A higher absolute zeta potential value indicates greater stability. Therefore, PCA demonstrated superior stability compared to PA and CA (Figure 1F), which can be attributed to the synergistic stabilizing effect of Pasp and CMC. The dual-polyelectrolyte system more effectively prevents premature transformation of ACP into crystalline calcium phosphate outside DTs, ensuring sustained release and long-term efficacy of mineralization precursors within DTs. This enhanced stability is crucial for achieving durable biomimetic mineralization.
Under neutral environmental conditions, we measured the concentrations of Ca²⁺ and PO₄³− released by PCA, PA, and CA after 24 h (Figure 1G). The results demonstrated that PA released significantly higher concentrations of Ca²⁺ and PO₄³− compared to the other two groups. Two primary reasons are proposed for this disparity. First, PA contains higher concentrations of Ca²⁺ and PO₄³− than the other two materials. Second, PA exhibits superior fluidity, facilitating easier ion dissociation and diffusion. Although PCA’s ion release concentration was lower than PA’s, it was markedly higher than CA’s. This difference may be attributed to the addition of Pasp in PCA, which enhances rheological properties, and whose carboxyl groups may promote calcium-phosphate ion release through chelation. Ion concentration analysis revealed that the Ca/P ratio remained stable at approximately 1.5, closely matching the characteristic ratio of ACP under neutral pH. This provides strong evidence for the presence of an ACP phase in the materials. Future studies should systematically compare the effects of different material states on ion release kinetics.
FTIR spectroscopy revealed the characteristic functional groups present in PCA, PA, CA, and PC. Specifically, PA exhibited a broad absorption band at 2700–3700 cm−¹, attributable to interactions between Ca²⁺ and hydroxyl (-OH) groups in Pasp. Peak shifts in the 1300–1600 cm−¹ region indicated electrostatic interactions between ACP and both amide (-CO-NH-) and carboxyl (-COOH) groups. Characteristic ACP peaks at 1065 cm−¹ (ν₃, asymmetric stretching) and 530 cm−¹ (ν₄, bending) confirmed binding between Pasp and ACP. The distinct PO₄³− vibrational modes at these wavenumbers provided direct spectroscopic evidence for successful Pasp-ACP complex formation (Figure 1H1).
For CA samples, the broad absorption peak in the 2700–3650 cm−¹ range originated from interactions between ACP and both -NH2 and -OH groups in CMC. In the 1300–1600 cm−¹ region, peak shifts with enhanced intensity suggested electrostatic interactions between Ca²⁺ and -COOH groups. Characteristic ACP peaks at 1054 cm−¹ (ν₃) and 560 cm−¹ (ν₄) further verified CMC-ACP binding. FTIR clearly demonstrated unique PO₄³− vibrational signatures at these positions, conclusively confirming effective CMC-ACP conjugation (Figure 1H2).
PC samples exhibited broad absorption in the 2700–3650 cm−¹ range, resulting from overlapping -OH/-NH groups of Pasp and electrostatic interactions with CMC’s -NH₂ groups. Peak shifts and increased intensity at 1300–1600 cm−¹ indicated interactions between the -COOH/-CO-NH- groups of Pasp and the -COOH groups of CMC. The peak at 1055 cm−¹ corresponded to the -C-O- stretching vibrations of secondary hydroxyl (-CH-OH) groups. After Pasp-CMC complexation, characteristic fingerprint peaks at 524 cm−¹ broadened (Figure S3).
PCA spectra showed broad absorption in the 2700–3700 cm−¹ region, primarily due to overlapping -OH/-NH groups from Pasp, electrostatic interactions with CMC’s -NH₂ groups, and interactions between ACP and the functional groups of both polymers. In the 1300–1600 cm−¹ range, peak shifts and enhanced intensity confirmed Ca²⁺-COOH electrostatic binding. The presence of ACP characteristic peaks at 1059 cm−¹ (ν₃) and 510 cm−¹ (ν₄) verified ternary complex formation. Distinct PO₄³− vibrational bands at these positions provided definitive evidence for Pasp-CMC-ACP ternary complex formation (Figure 1H3).
As biomimetic analogs of NCPs, polyelectrolytes synergistically guide the ordered deposition of ACP within collagen fibers. Comparative studies using monolayer reconstituted collagen fiber models revealed that PCA, PA, and CA exhibit different mineralization capacities (Figure 2). Combined TEM and SAED analyses demonstrated that after 1 day of incubation, weak arc-shaped diffraction at the (002) crystal plane appeared in the PCA- and PA-treated groups (Figure 2A1 and B1), whereas no significant diffraction signals were detected in the CA group (Figure 2C1). By day 3, the PCA group displayed clear arc-shaped diffraction at both (002) and (211) crystal planes (Figure 2A2), indicating the orderly alignment of HAP crystals along the collagen c-axis, thereby achieving typical intrafibrillar mineralization. In contrast, the PA group showed only (002) plane diffraction (Figure 2B2), whereas the CA group required until day 5 to exhibit weak signals (Figure 2C3). Ultimately, the PCA group achieved complete mineralization (Figure 2A3), outperforming the partial mineralization observed in the PA group (Figure 2B3) and the limited mineralization of the CA group (Figure 2C3). All experimental groups exhibited interfibrillar needle-like crystals, confirming the presence of extrafibrillar mineralization, whereas no mineralization was detected in the PC group throughout (Figure 2D1–D3).
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Figure 3 (A) Dentin sensitivity models were made and subsequently grouped to treat dentin discs for biomimetic remineralization and the occlusion of DTs. Friction and acid immersion challenges were carried out following 28 days of treatment. SEM images of the (B1) dentin surfaces, (B2) longitudinal sections, and images (B3) post-friction and (B4) acid immersion in the blank group after 28 days of treatment. (C–G) SEM images of the other five groups.
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At the molecular level, Pasp facilitates ACP infiltration into fibers via the polymer-induced liquid-precursor (PILP) process.43–46 The carboxyl groups on its molecular chains may regulate mineralization by interacting with specific collagen sites (eg, -OH, -NH₂),47 although precise binding sites require further elucidation using techniques such as atomic force microscopy (AFM). CMC influences mineralization through several molecular interactions: its carboxymethyl groups may provide nucleation sites via electrostatic interactions with collagen -NH₂, and molecular forces between -NH₂ and collagen -COOH;48 concurrently, CMC alters collagen self-assembly kinetics, which may explain its mineralization-promoting mechanism at the material-extracellular matrix interface.34
The PCA system demonstrates superior mineralization performance, primarily due to the synergistic effects of Pasp and CMC. Pasp ensures stable ACP infiltration, whereas CMC optimizes the collagen matrix structure and supplies additional nucleation sites. This dual action enables PCA to exceed PA and CA in both mineralization rate and completeness. Notably, the PC group results confirm that Pasp and CMC alone cannot initiate effective mineralization in the absence of ACP, highlighting ACP’s essential role in biomimetic mineralization. Although this study provides morphological and structural validation of PCA’s mineralization advantages, subsequent mechanical testing, such as nanoindentation, will be necessary to comprehensively evaluate the mechanical enhancement of mineralized collagen, thereby providing a stronger scientific basis for clinical translation.
Through systematic comparison of the mineralization capabilities of four recombinant collagen fibers (PCA, PA, CA, and PC), this study further evaluated their effects on dentin remineralization and DT occlusion through in vitro experiments (Figure 3A). After establishing DH models (see Preparation of DH models), SEM revealed differences in mineralization outcomes among groups after 28 days of treatment.
In the blank group, collagen fibers on the dentin surface remained exposed, with only minimal mineral deposition observed within the tubules and no evidence of mineral penetration into deeper DT regions (Figure 3B1 and B2). The Gluma group exhibited partial surface coverage and a mineral penetration depth of approximately 12 μm, with occasional tubules showing mineral deposits reaching 20–30 μm (Figure 3C1 and C2). The PCA and PA groups showed the most robust surface occlusion effects, achieving complete collagen fiber coverage and intratubular mineralization (Figure 3D1 and E1). The PCA group displayed an average penetration depth of 30 μm (Figure 3D2), whereas the PA group averaged 15 μm, with some DTs exhibiting mineral penetration up to 39 μm (Figure 3E2). The CA group achieved only partial fiber coverage and incomplete tubule occlusion, with a penetration depth of about 15 μm (Figure 3F1 and F2). The PC group, whereas able to cover surface fibers, showed sparse intratubular mineralization with a penetration depth of approximately 5 μm (Figure 3G1 and G2).
In mechanical friction and acid-etching challenge tests, the groups exhibited distinct stability characteristics. The blank group, with initially low mineralization, showed no change in mineral content after treatment (Figure 3B3 and B4). The Gluma group displayed significant mineral loss and fiber re-exposure after acid etching (Figure 3C4). The PCA and PA groups demonstrated exceptional stability, maintaining consistent mineral content after both challenges (Figure 3D3, D4, E3, and E4). The CA group resisted mechanical friction but failed to withstand acid etching (Figure 3F3 and F4), whereas the PC group lost mineral under both challenges (Figure 3G3 and G4). These differences may result from variations in the crystalline structure of the mineralization products.
EDS analysis revealed that the PCA group had a Ca/P ratio of 1.66, closest to the theoretical value of HAP (1.67), indicating successful transformation of ACP into the crystalline form. The PA group (Ca/P ratio: 1.75) and CA group (Ca/P ratio: 1.79) showed slight deviations, suggesting the coexistence of other calcium phosphate phases. In contrast, the abnormally high Ca/P ratio of 2.1 in the PC group confirmed its failure to achieve biomimetic mineralization conversion (Figure 4A).
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Figure 4 (A) Contents of Ca and P in demineralized dentin after incubation with PCA, PA, CA, and PC for 28 days. (B) Dentin airtightness data for each group following 28 days of treatment and after friction and acid immersion. Values with different letters indicate significant differences (p < 0.05). (C) Dentin hardness data for each group after 28 days of treatment. Values with different letters indicate significant differences (p < 0.05).
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Dentin primarily consists of organic components such as collagen and NCPs.49 At the molecular level, Pasp, containing aspartic acid residues as an NCP analog, promotes remineralization through a dual mechanism: (1) its acidic domain stabilizes ACP and accelerates its conversion to HAP, reducing the remineralization cycle from 7 days to 2 days;50 and (2) it specifically recognizes collagen active sites to form mineralization nucleation centers.47 Capillary effects further facilitate deep penetration into DTs for physical occlusion. CMC also exhibits NCP-mimicking functionality, but its poor flow properties limit penetration depth, explaining why Pasp-containing PA outperformed CMC-containing CA in occlusion depth. Notably, the superior resistance to friction and acid etching observed in the PCA and PA groups confirms a correlation between mineralization quality and clinical durability.
This study selected Gluma as a positive control to reflect the occlusion efficacy of clinically effective materials. However, its protein precipitation mechanism differs from the biomimetic mineralization approach used here. Future studies will incorporate additional control groups for more comprehensive comparisons, including positive controls for remineralization mechanisms. Optimizing the fluidity of CMC may also enable deeper and more durable tubule occlusion.
The study optimized a dentin sealing test device, employing an air pressure measurement method to evaluate dentin sealing ability and DT permeability, whereas comparing the sealing efficacy of different treatment groups. After 28 days of treatment, followed by acid immersion and abrasion, dentin sealing ability was assessed. The PCA group showed no significant difference in dentin sealing ability compared to normal dentin (p ≥ 0.05), and its performance remained stable after acid immersion and abrasion, indicating that PCA effectively restored dentin sealing (Figure 4B). In contrast, the PA group exhibited significantly lower sealing ability than normal dentin (p < 0.05), but no further decline was observed after acid immersion and abrasion. The blank control, Gluma, CA, and PC groups demonstrated improved sealing compared to the demineralized group, yet all remained significantly lower than normal dentin (p < 0.05). Furthermore, their performance declined further after acid immersion and abrasion, suggesting limited efficacy in restoring dentin sealing. These test results aligned with the findings for remineralization and tubule occlusion, confirming that PCA effectively restored the structural integrity of demineralized dentin.
For mechanical properties, Vickers hardness testing showed that dentin hardness in the PCA and PA groups was significantly higher than that of the demineralized group (p < 0.05) and did not differ significantly from normal dentin (p ≥ 0.05), indicating effective restoration of dentin mechanical performance (Figure 4C). The Gluma and CA groups exhibited higher hardness than the demineralized group, but values remained significantly lower than those of normal dentin (p < 0.05), with no significant difference between Gluma and CA (p ≥ 0.05), suggesting limited restorative effects. The blank control and PC groups showed no significant difference in hardness compared to the demineralized group (p ≥ 0.05) and were significantly lower than all other treatment groups (p < 0.05), indicating a lack of effective restoration of dentin mechanical properties.
These comprehensive results demonstrate that PCA and PA not only provide durable DT occlusion and significantly enhance sealing but also effectively improve dentin mechanical properties, making the tissue more resistant to mechanical and chemical stimuli in the oral environment. This dual mechanism supports both the treatment and prevention of DH recurrence. In contrast, CA showed efficacy comparable to that of Gluma, a commonly used clinical desensitizer, whereas PC lacking ACP produced almost no noticeable therapeutic effect.
Dental plaque biofilm formation is closely associated with the initiation and progression of dental caries, with S. mutans recognized as a major pathogenic bacterium due to its strong acidogenicity, acid tolerance, and capacity for extracellular polysaccharide synthesis.51 In this study, S. mutans was anaerobically cultured for 36 h, followed by treatment with deionized water (control), PCA, CA, or PC for an additional 24 h to systematically evaluate effects on plaque biofilm. CLSM revealed that the dentin surface in the control group was fully covered by viable bacteria (green fluorescence), whereas the PCA, CA, and PC groups exhibited significantly reduced viable bacterial coverage (p < 0.05) and increased proportions of dead bacteria (red fluorescence) (p < 0.05) (Figure 5A and B). No significant difference in total biofilm biomass was observed among the PCA, CA, and PC groups (p ≥ 0.05) (Figure 5C). SEM confirmed reduced S. mutans adhesion on dentin surfaces in the treatment groups (Figure 5D). Colony-forming unit (CFU) counts showed that, after 12 and 24 h of incubation, bacterial counts in the PCA, CA, and PC groups were significantly lower than in the control group (p < 0.05) (Figure 5E and F). These findings indicate that PCA, CA, and PC effectively inhibit S. mutans biofilm formation and demonstrate bactericidal activity.
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Figure 5 (A) CLSM images of live/dead bacterial staining for the Control, PCA, CA, and PC groups. Live bacteria were stained green with DMAO, while dead bacteria were stained red with PI. (B) The percentage of live/dead bacteria in each group (** p < 0.01). (C) The percentage of live bacteria relative to the total number of bacteria in each group. Values with different letters indicate significant differences (p < 0.05). (D) SEM images of bacteria adhering to the surface of dentin discs in each group. (E) Representative photographs of each group of S. mutans colonies grown on BHI agar plates. (F) CFU counts. Values with different letters indicate significant differences (p < 0.05).
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The antibacterial mechanism of CMC likely operates through multiple pathways.52,53 Positively charged CMC molecules disrupt bacterial membrane integrity via electrostatic interactions with phospholipids, inhibit key metabolic enzymes (eg, respiratory and synthetase enzymes), and interfere with genetic replication by binding to nucleic acids. CMC may also activate bacterial autolysin systems or apoptotic pathways. Notably, although PC was expected to demonstrate superior antibacterial efficacy due to its higher CMC content, the experimental data showed no significant difference among the three groups. This observation may be explained by the synergistic effects of Ca²⁺ and PO₄³− released by PCA and CA: Ca²⁺ destabilizes bacterial membranes by binding to anionic components, whereas PO₄³− modulates the microenvironmental pH to suppress bacterial growth. PCA and CA may also provide sustained ion release at the bacterial surface, enhancing antibacterial persistence.
Regarding experimental design, a blank control was used to minimize interference from the culture medium and environmental factors. Subsequent investigations will prioritize comparisons with existing commercial products to assess clinical applicability. Additionally, future studies will include synergistic antibacterial agents and develop multispecies biofilm models to better mimic the oral microecological environment, thereby optimizing antibacterial efficacy.
To evaluate the cytotoxicity of PCA, PA, CA, and PC, HDPSCs were successfully isolated and cultured from extracted teeth. The isolated cells displayed typical spindle-shaped morphology (Figure S4) and, as confirmed by flow cytometry, met the criteria of HDPSCs: high expression of mesenchymal stem cell markers (CD73, CD90, and CD95, all > 95% positivity) and low expression of hematopoietic lineage markers (CD34 and CD45, both < 2% positivity) (Figure S5).
Live/dead fluorescence staining showed good cell viability across the PCA, PA, CA, and PC groups, with no significant differences in the proportions of live or dead cells compared to the control (p ≥ 0.05) (Figure 6A and B). The CCK-8 assay further confirmed that HDPSC viability in all groups did not differ significantly from the control (p ≥ 0.05) (Figure 6C), indicating no apparent inhibitory effect on cell growth. Additionally, hemolysis testing revealed that hemolysis rates for all groups were below 5% (Figure 6D and E), meeting established safety standards for biomaterials. These results collectively demonstrate that PCA, PA, CA, and PC exhibit good cytocompatibility and hemocompatibility.
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Figure 6 (A) CLSM images of live/dead cell staining for the Control, PCA, PA, CA, and PC groups. Live cells were stained green with Calcein AM, while dead cells were stained red with PI. (B) The percentage of live/dead cells in each group (ns = not significant). (C) CCK-8 results for HDPSCs in each group over 1, 3, and 5 days (ns = not significant). (D) Representative photographs of the hemolysis experiments in each group. (E) The percentage of the hemolysis rate in each group.
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CMC, as a polyelectrolyte, serves multiple roles in regulating HDPSC behavior. Studies show that CMC, when combined with nano-hydroxyapatite (nHAP) to form a composite scaffold, not only promotes HDPSC adhesion and proliferation but also significantly upregulates the expression of odontogenic markers such as DSPP, dentin matrix protein-1 (DMP-1), alkaline phosphatase (ALP), and vascular endothelial growth factor (VEGF), thereby enhancing mineralization and tissue regeneration.54,55 This may occur via a polyelectrolyte-induced “cell aggregation phenomenon”, in which cells form high-density aggregates through electrostatic interactions within the CMC microenvironment, enhancing intercellular communication and differentiation synergy. Future research should investigate the association between polyelectrolyte charge density and the spatiotemporal dynamics of cell aggregation to optimize biomimetic mineralization strategies.
PCA demonstrates unique advantages in treating DH and caries prevention. Its biomimetic remineralization mechanism forms mineral structures closely resembling natural HAP, effectively occluding DTs and repairing demineralized tooth structures, thereby providing durable therapeutic effects. Experimental data confirm that PCA significantly promotes dentin remineralization and exhibits antibacterial properties that inhibit cariogenic bacterial growth, achieving both DH relief and caries prevention. Compared with conventional desensitizing agents, PCA shows long-term efficacy. And PCA has superior biocompatibility. To further validate clinical applicability, future studies will employ experimental models that better simulate actual oral conditions, including extended mechanical-chemical challenge cycles, multi-species biofilm systems, and dynamic simulation for functional assessment, as well as animal experiments under near-physiological conditions. These improvements will support more accurate prediction of PCA’s performance in complex oral environments, providing stronger evidence for clinical application.
Conclusion
PCA emerges as a promising multifunctional agent for dental applications. Its ability to effectively promote dentin remineralization and occlude DTs positions it as a viable solution for the treatment and prevention of DH. Additionally, PCA’s inherent antimicrobial properties contribute to caries prevention by inhibiting bacterial growth. These dual benefits—biomimetic mineralization and antibacterial activity—highlight PCA’s potential as a superior alternative to conventional desensitizing agents. However, further in vivo studies and long-term clinical evaluations are required to validate its safety, stability, and practical utility. Future research could also explore synergistic combinations with other bioactive materials to enhance therapeutic efficacy.
Ethics Approval and Informed Consent
This study was conducted in accordance with the principles of the Declaration of Helsinki. The Medical Ethics Committee of the Affiliated Hospital of Qingdao University approved the study protocol (QYFYWZLL29632). Informed consent has been obtained from the donors of the extracted teeth and blood samples.
Acknowledgments
The study was funded by grants from the Youth Innovation Team Program of Universities in Shandong Province (2022KJ297).
Disclosure
The authors declare that this study is not associated with any conflicts of interest.
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