Peptide self-assembly property analysisPeptide structure analysis

To investigate the structural modulatory effect of the GKG motif on peptides, α-helical peptides ranging from 11 to 37 amino acids were selected (Table S1).38,39,40,41,42 Both the α-helical monomers and the helix-GKG-helix constructs were first subjected to structural prediction using AlphaFold 3.43 Subsequent structural visualization and analysis were performed with ChimeraX 1.10.1.44

Peptide synthesis

Peptides TKH (TKRQQVVGLLWHLLHHLLHGKGTKRQQVVGLLWHLLHHLLH), TVH-19 (TKRQQVVGLLWHLLHHLLH) were synthesized through solid-phase Fmoc-based chemistry by Bintuo Biotechnology Co., Ltd. (Hangzhou, China). The synthesized peptides were subsequently purified (>90%) by high-performance liquid chromatography and stored at −20 °C.

Circular dichroism (CD)

CD spectra were obtained in the range of 190–260 nm at room temperature using a CD spectrometer (Chirascan, Applied photo physics, England). The peptide TKH solution was diluted with 4.2 mmol/L PBS, 25 mmol/L SDS buffer (pH 7.0) to final concentration at 200 μmol/L. The measurements were conducted after 24 h of incubation at 37 °C. All spectra were obtained using a 2 nm bandwidth and a 1 nm/s scanning rate and were normalized to mean residual ellipticity values.

Fluorescence recovery after photobleaching (FRAP)

The TKH peptide solution (5 mg/mL) was prepared in 10 mmol/L PBS buffer (pH 7.4). The peptide was labeled with the fluorescent dye Rh6G (Solarbio, IR2030) at a final concentration of 10 μmol/L, followed by incubation in a shaking incubator at 37 °C and 200 r/min for 1–2 h.32 After sonication for 10 min, the sample was applied onto a glass slide and allowed to equilibrate in the dark for 15 min. TKH solution (5 mg/mL) and 10 μmol/L Rh6G were prepared in parallel as the control. Droplet formation was observed using confocal laser scanning microscope (CLSM, Olympus FV3000, Japan). A circular region with a radius of 1 μm was photobleached using a 555 nm laser at 100% intensity with 10 iterations. Fluorescence recovery in the bleached area was subsequently monitored using the same wavelength. Data acquisition was performed using Zen Blue software (Zeiss). For data normalization, the pre-bleach fluorescence intensity was set as 100%, while the first post-bleach measurement was set as 0%. The relative fluorescence recovery rate was calculated according to the equation\(\,_}\left(t\right)=\frac_}}_-}-_}}\). The final FRAP recovery curve represents the average of recovery curves obtained from three independent samples.

Phase diagram construction via turbidity assay

Buffer systems with pH values of 5.5, 6.0, 6.5, 7.0, and 7.5 were prepared using 20 mmol/L Tris, 0.2 mol/L NaCl, and 10 mmol/L PBS. TKH peptide solutions were then prepared in these buffers at final concentrations of 0.1, 0.2, 0.3, 0.4 mmol/L. The turbidity of each solution was measured in triplicate (n = 3) using a 96-well plate at λ = 350 nm under varying pH and concentration conditions. The phase transition pH corresponding to different concentrations were plotted on a concentration–pH coordinate system to generate experimental data sets.45

Transmission electron microscopy (TEM)

TKH and TVH-19 peptides were dissolved in 10 mmol/L phosphate-buffered saline (PBS, pH 7.4) to a final concentration of 200 μmol/L and allowed to equilibrate at room temperature for 2 h. The peptide solutions were then deposited onto carbon-coated copper grids. After the samples were dried. high-resolution TEM images were acquired using transmission electron microscope (JEM-2100 Plus, JEOL, Japan) operated at an accelerating voltage of 200 kV.

Thioflavin T (ThT) spectroscopy assay

TKH/TVH-19 peptide solutions with concentrations ranging from 0 to 100 μmol/L were prepared in PBS and mixed with a 10 μg/mL ThT solution. After incubation at room temperature for 10 min, the fluorescence intensity was measured using an Infinite M200 PRO plate reader (Tecan) with λex = 438 nm and λem = 495 nm. Similarly, different concentrations peptide solutions were prepared, and the ThT fluorescence intensity was measured with λex = 380 nm and λem = 400–580 nm. The fluorescence values were normalized to the ThT fluorescence intensity of the PBS group. Each sample was done in triplicate.

Tyndall effect detection

The Tyndall effect of different concentrations of TKH (0–200 μmol/L) and TVH-19 (0–400 μmol/L) prepared with 10 mmol/L PBS was detected by shedding light on the tubules using a small laser light.

Antibacterial properties of self-assembly peptidesCrystal violet assay

48-well plates were pre-coated with human saliva (37 °C, 2 h) and UV-sterilized to form a mature acquired pellicle. Biofilms of S. mutans (1 × 10⁴ CFU/mL in BHIS) were grown anaerobically for 24 h in the presence of TKH or TVH-19 peptides (2.5–40 μmol/L, prepared with DDW). After incubation, biofilms were washed, fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet. Biofilm formation was qualitatively assessed by microscopy (OLYMPUS SZX16) and quantified by measuring the OD595 of the dissolved dye after elution with 33% glacial acetic acid.

pH drop assay

The pH drop assay was used to evaluate the peptide effects on metabolic activity of S. mutans biofilms. Acquired pellicle-coated 12-well plates were inoculated with 1 mL of S. mutans suspension (1 × 104 CFU/mL) in BHIS medium and incubated anaerobically for 24 h to form biofilms. After removing the supernatant, biofilms were gently rinsed three times with PBS to remove residual medium. Subsequently, 2 mL of 10 mmol/L glucose solution (PBS-based, pH = 7.0) containing different concentrations of TKH/TVH-19 peptides was added to each well. The pH changes were monitored and recorded at 30-min interval.

Demineralized dentin specimen preparation

Approved by the Ethics Review Committee of West China School of Stomatology (WCHSIRB-D-2022-117), the extracted caries-free human third molars were gathered at West China Hospital of Stomatology and stored in the PBS solution containing 0.5% thymol at 4 °C. Dentin disks (3.50 mm ± 0.50 mm thick) were sectioned from the crowns near the cementoenamel junction using a water-cooled, low-speed diamond saw (EXAKT, Germany) and embedded in acrylic resin. The surfaces were then polished sequentially with 800- to 3 000-grit carbide paper (Struers, Denmark) and ultrasonically cleaned for 5 min to remove the smear layer. Specimens with a Vickers microhardness (25-gf load, 10 s dwell time) between 0.60 and 0.80 GPa, determined from five indentations per disk, were selected. A 4 × 4 mm² window was exposed on each specimen’s surface by coating the surrounding area with acid-resistant nail varnish. Demineralization was performed by immersing the specimens in 0.5 mol/L EDTA (pH 8.0) for 30 min. Finally, the demineralized dentin specimens were ultrasonically cleaned and stored in PBS at 4 °C until use.

Characterization of the binding capacity of self-assembly peptidesIsothermal titration calorimetry (ITC)

ITC was performed to quantify the thermodynamic parameters of the interaction between TKH peptide and type I collagen using a Nano ITC microcalorimeter (TA Instruments, USA) at 25 °C. All samples were dissolved in the same buffer (20 mmol/L HEPES, 150 mmol/L NaCl, pH 7.4) and degassed under vacuum for 10 min prior to use. The TKH peptide solution (1 μmol/L) was loaded into the sample cell, while the type I collagen solution (10 μmol/L) was placed in the titration syringe. A total of 20 injections (2 μL each) were performed at 200 s intervals with a stirring speed of 300 r/min. The heat flow (μJ/s) versus time was recorded, integrated, and corrected for baseline to obtain the heat released per injection. The resulting binding isotherm was fitted using a one-site binding model in NanoAnalyze software (TA Instruments) to calculate thermodynamic parameters (Kd is the dissociation constant, N is the number of TKH that bind to a single collagen molecule). Based on the equation ΔG = RTlnKd = ΔH − TΔS, where R is the gas constant, T is the absolute temperature (in Kelvin), the values of ΔG and ΔH can thus be determined.46

Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) analysis

The demineralized dentin specimens were coated using 100 μL of TKH (100 μmol/L) and TVH-19 (200 μmol/L) solution (prepared with 10 mmol/L PBS) and air-dried at room temperature after 1 h treatment. The FTIR spectra of demineralized dentin specimens, TKH/TVH-19-coated dentin specimens and dentin specimens rinsed with DDW after coating were obtained by ATR-FTIR analysis (Nicolet i50; Thermo Scientific, USA).

The bicinchoninic acid (BCA) assay

The BCA method was used to test the adsorption amount of TKH/TVH-19 on HA particles (Sigma-Aldrich, USA). 200 μL TKH/TVH-19 peptide solutions (0.5 mg/mL, 1 mg/mL, prepared with DDW) were mixed with or without HA particles (20 mg per tube) in each group (n = 3).47 Tubes were placed at room temperature for 12 h for complete reaction. Tubes containing the peptide solution and HA were centrifuged (13,000 rpm, 3 min) and the supernatant was collected to detect the unbound peptide concentration using the Enhanced BCA Protein Assay Kit (Beyotime).

CaP stabilization assay

Mineralization media containing 4.5 mmol/L CaCl₂, 2.1 mmol/L Na₂HPO₄, and varying concentrations of TKH (3.125–200 μmol/L) or TVH-19 (6.25–400 μmol/L) peptides were prepared in DDW by sequential addition of phosphate and peptide stocks to the calcium stock. Following a 24 h incubation at 37 °C, turbidity was visually assessed. The precipitates obtained by centrifugation (4 000 r/min, 5 min) were microscopically examined for calcium phosphate crystals.

Collagen mineralization

Type I collagen from rat tails (Sigma-Aldrich, C7661) was dissolved in 0.1 mol/L acetic acid to prepare a stock solution (3 mg/mL) and stored at 4 °C. For collagen reconstitution, a glycine buffer (50 mmol/L glycine, 200 mmol/L KCl, pH 9.2) was freshly prepared. A working solution (50 μg/mL) was obtained by mixing 16.7 μL of the collagen stock with 983.3 μL of the glycine buffer, followed by vortexing and incubation at room temperature for 20 min.48 Subsequently, 14 μL aliquots of the reconstituted collagen solution were applied to nickel grids placed in a six-well plate. Humidity was maintained by adding DDW to the vacant wells. The plate was sealed and incubated at 37 °C for 24 h. The grids were then washed with DDW, fixed on droplets of 0.5 wt% glutaraldehyde (200 μL) for 2 h, washed again, and air-dried. For mineralization, the collagen-coated grids were incubated at 37 °C for 5 days in a humidified chamber while floating on one of the following solutions: 200 μg/mL PAH (positive control), 100 μmol/L TKH, 200 μmol/L TVH-19, or DDW (negative control). Finally, the grids were rinsed with DDW, air-dried, and examined by transmission electron microscopy (JEM-2100 Plus, JEOL, Japan).

Dentin remineralization in vitro

The demineralized dentin samples were randomly divided into 4 groups (n = 15), and the window surfaces were treated with the following agents: the negative control group: PBS; the positive control group: 1 000×10-6 NaF; the peptide groups: TKH (100 μmol/L), TVH-19 (200 μmol/L). Each specimen was exposed to the above-mentioned solutions for 1 h once a day. Then, they were rinsed three times with DDW, followed by immersion in artificial saliva (AS) (0.5 mmol/L CaCl2, 0.9 mmol/L KH2PO4, 130 mmol/L KCl, 20 mmol/L HEPES, pH 7.0, each specimen for 7 mL) in a 37 °C incubator for 4 weeks.49 The AS (pH 7.0) was freshly prepared daily.

SEM and EDS mapping analysis

The dentin specimens were immersed in 2.5% glutaraldehyde for 4 h at room temperature, rinsed 3 times with PBS solution, and then dehydrated with gradients of ethanol in different volume fractions (30, 50, 70, 90, 100%) for 30 min each time. The specimens were sprayed with gold, and the structures and compositions of the occlusal and longitudinal sections of the dentin specimens were observed by SEM (Apreo S, FEI, Eindhoven, The Netherlands). The content and distribution of calcium (Ca) and phosphorus (P) were determined by EDS elemental mapping.

X-ray diffraction (XRD) analysis

After 4 weeks, specimens of four groups were removed from AS and air dried for XRD (Ultima IV, Rigaku, Japan) analyses. At the same time, sound dentin and demineralized dentin disks were used as controls.

Dentin remineralization in vivo

All animal experiments complied with the Chinese Council on Animal Care guidelines and received ethical approval from the West China College of Stomatology Animal Experimentation Committee (WCHSIRB-D-2022-215). Human dentin specimens were processed into oval-shaped slices (4 mm diameter × 2 mm thickness) with smooth edge (Fig. 5b). The samples were demineralized by etching with 37% phosphoric acid for 15–20 s, followed by ultrasonic cleaning for 15 min. Two holes were symmetrically drilled on both sides of each slice to facilitate fixation in the rat oral cavity. Specific pathogen-free Sprague-Dawley rats (6-8 weeks old, 200-300 g body weight, n = 8 for each group, Chengdu Dashuo Biotechnology Co., Ltd.)) were selected for the study. A 0.50 mm stainless steel ligature wire was inserted through the interproximal space between the first and second molar and anchored to the anterior teeth, securing the dentin sample to the rat palate.9,50 Excess wire was trimmed, and sharp edges were coated with resin to prevent mucosal injury. Rats were randomly divided into four groups and treated daily for 28 days with: PBS (negative control), 1 000 ppm NaF (positive control), 100 μmol/L TKH, or 200 μmol/L TVH-19 (100 μL applied locally). Body weight was monitored, and a soft diet was provided with water withheld for 1 h post-treatment. After 28 days, dentin samples were collected for SEM/EDS analysis. Major organs and oral tissues were harvested for histological examination (H&E staining), and blood samples were taken for hematological analysis.

Molecular dynamics (MD) simulation of self-assembly peptides on dentin adsorption and remineralization

The initial structures of peptides TKH and TVH-19 were predicted using AlphaFold 3. Since HAP is the primary inorganic component of dentin and the acid-etched dentin surface predominantly exposes the (010) crystal plane, the HAP (010) surface was selected as the target model.51 The HAP surface model was constructed using CHARMM-GUI, after which TKH and TVH-19 peptides were individually placed on the HAP surface to generate the initial adsorption configurations (Figure S6). MD simulations were performed using GROMACS 2019.1, with two systems established: (1) the peptide–HA adsorption system; (2) the peptide–HA remineralization system. In the remineralization simulations, ions were added according to the experimental remineralization solution (2.58 mmol/L CaCl₂·2H₂O, 1.55 mmol/L KH₂PO₄, 10 mg/L NaF, 180 mmol/L NaCl, and 50 mmol/L Tris–HCl, pH 7.6).52 Specifically, 65 Ca²⁺, 40 H₂PO₄−, and 6 F− ions were inserted into the equilibrated adsorption structure, and additional Cl⁻ ions were introduced to neutralize the system. This setup ensured consistency between the simulated ionic environment and the actual remineralization conditions. Energy minimization was performed using the steepest descent method, followed by equilibration under NVT and NPT ensembles. Temperature was controlled at 298 K via the V-rescale thermostat, and semi-isotropic pressure coupling was maintained at 1 bar (Z direction) and 0 bar (X/Y direction) using the Parrinello–Rahman barostat. The production simulations for both adsorption and remineralization systems were each run for 100 ns. Interaction energies between peptides and (i) HAP surface, (ii) Ca²⁺ ions, and (iii) H₂PO₄− ions were computed using GROMACS energy modules, and the average binding energies were obtained from the equilibrated trajectories. Structural stability and conformational changes were analyzed through RMSD, RMSF, and DSSP secondary structure calculations. Visualization and data analysis were performed using Pymol and Matplotlib, respectively.

Statistical analysis

Each experiment was repeated at least 3 times. All results are presented as the mean ± standard deviation (SD). GraphPad Prism 9.5 and Origin 2024 software were used to perform statistical analyses. Statistical analyses between two groups were performed using Student’s t-tests. Differences among multiple groups were evaluated using one-way ANOVA and Dunnett’s multiple comparison test. Statistical significance was accepted at P < 0.05.