Dental caries is a common oral disease that seriously threatens oral health. It is mainly caused by acid erosion and demineralization of enamel, resulting in the loss of minerals under the surface layer. Enamel remineralization is the key to preventing and treating dental caries. Among them, enamel protein Ameloginin plays an important role in remineralization. It can regulate crystal growth and arrangement and thus promote remineralization. The current remineralization systems are mainly based on enamel amelogenin proteins (such as leucine-rich amelogenin peptide, amelogenin derivatives), or dentin phosphoprotein (DPP), and synthetic peptides similar to salivary proteins (such as P11-4).

Hydrogels, a biopolymer with unique characteristics, are widely used in dental repair and tissue regeneration. Hydrogels with appropriate encapsulated components can enhance teeth' antibacterial and remineralization abilities and show excellent application potential in the treatment of dental caries. However, hydrogels still face challenges in aspects such as design, material preparation, and dentin repair. Therefore, the combination of bioactive hydrogels and peptides has become a research hotspot.

Recently, Codruta Sarosi et al. from Romania published an article titled "Developing Bioactive Hydrogels with Peptides for Dental Application" in Biomedicines. This study focuses on evaluating the self-assembled microstructure and network behavior of newly developed bioactive hydrogels rich in leucine amelogenin peptide (LRAP), providing new ideas and methods for enamel remineralization research.

Materials and Methods

Peptides corresponding to porcine amelogenin P173 were manufactured by Synpeptide Co., Ltd., purified and identified by HPLC and ESI-MS, and then made into a stock solution. Hydrogels were prepared by mixing PEG400, fumed silica nanoparticles, distilled water, hydroxyapatite, and adding 2-Hydroxyethyl salicylate. The obtained hydrogel was divided into three parts: two with peptides P1 and P2 added respectively, and one as control G0. Experimental hydrogel G3 had the same components as the others plus nanocapsules with P1 peptide. The hydrogels were analyzed by FTIR spectrometer (wavenumber range 4000–400 cm⁻¹ using ATR technique) and UV-Vis spectrophotometer (measurement range 400–1000 nm). For the antibacterial test, three streptococci were cultured on media, diluted, and inoculated into Petri dishes with test and control gels to observe inhibition zones. For the cytotoxicity test, hydrogels were co-cultured with HFL1 cells using a co-culture technique, and cell viability was determined by MTT method with one-way ANOVA. The hydrogels were observed by SEM and AFM microscopes to analyze surface roughness parameters.

Results

Firstly, they investigated the obtained hydrogels through FTIR spectroscopy to provide data for the secondary structure characterization of the peptides. The FTIR spectra of peptides and hydrogels with peptides resembled that of bovine pancreatic trypsin inhibitor. The FTIR-ATR spectra highlighted the presence of peptides at specific bands. In G0, G1, and G2 spectra, absorption bands were associated with the PO₄³⁻ group from hydroxyapatite, and the peak at 1462 cm⁻¹ was related to the CO₃²⁻ group. It may suggest that carbon from the organics does not pyrolyze completely and may instead dissolve into a HA crystal.

Fig. 1 The FTIR spectra of investigated hydrogels.

Next, they used UV absorption spectroscopy on experimental hydrogels to study the secondary and tertiary structure of peptides as well as their association behavior. It was found that G1 and G3 hydrogels containing P1 peptide had an absorption peak at 219 nm, with the intensity of this peak increasing compared to G2 without P1. G0 had a low-intensity absorption band at 219 nm. G1 had an absorption peak at 269 nm and G3 had a less intense absorption peak at 259 nm.

Fig. 2 UV-Vis spectra of experimental hydrogels.

To determine the antibacterial effect of different hydrogels, Tukey's test of antibacterial activity against S.mutans, S.salivarius, and S.thermophillus was performed. After incubating samples at 37℃, the inhibition zones of tested microbial strains were measured. The control sample G0 showed no inhibition. For S. mutans strains, there were significant differences among gels. G1 and G3 (with P1) had high inhibition for Streptococcus mutans, while G2 (with P2) had low inhibition. G3 (with P1) had good inhibition for Streptococcus salivarius and the highest inhibition (13 mm) for Streptococcus thermophilus. G1 and G2 had low inhibition for this strain. The addition of nanocapsules with P1 peptide enhanced the antibacterial effect of hydrogel G3. Cytotoxicity tests showed no significant differences between the gels.

Fig. 3 Tukey's test of antibacterial activity of experimental hydrogels.

SEM microscopy was used to observe the general aspect of deposited thin films. G0 contained only nanostructured hydroxyapatite and silica, forming island structures with minor cracks. Adding peptide 1 in G1 increased island sizes and border cracks and formed microstructural clusters. Peptide 2 in G2 was effective in spreading, generating large uniform domains, and G3 contained small microcapsules. The fine microstructure of the thin films was also analyzed. Peptide 1 and peptide 2 had different effects on the microstructure, and G3 samples had microcapsules embedded in a uniform and compact structure. More enhanced microscopic techniques were required for their proper observation.

Fig. 4 SEM images of the experimental hydrogels' general aspect.

Then, they aimed to analyze the different hydrogel samples using Atomic Force Microscopy (AFM). G0 sample topography showed dense agglomeration of submicron clusters. Peptide 1 in G1 facilitated fine clusters' reticulation. Peptide 2 in G2 enhanced the reticulation effect. The G3 sample had small microspheres that facilitated the uniform distribution of mineral filler. Peptide 1 and peptide 2 improved the nanostructure, and chitosan mediation of peptide 1 had a nanostructural synergy in G3.

Fig. 5 AFM topographic images of the experimental hydrogels' fine microstructure.

The addition of peptides decreased roughness at the fine microstructure level, while the G3 sample had slightly increased roughness due to remaining microcapsules. Additionally, the peptide reticulation facilitated the uniformity of thin film nanostructure.

Fig. 6 Surface roughness values measured at a scanning area of (a) 20 μm × 20 μm and (b) 5 μm × 5 μm.

Conclusion

In conclusion, this research aimed to develop hydrogels with peptides for enamel remineralization. The study observed the influence of peptides through cell culture and microbial strains, using techniques like FTIR, UV-Vis spectroscopy, SEM, and AFM. The results are promising for enamel repair and microbial control, especially for Streptococcus mutans, but practical applications require further research and validation. Future studies are needed to demonstrate remineralizing properties and address challenges such as determining optimal component ratios.

• Anti-Amelogenin, X Isoform
• Human Amelogenin, X Isoform ELISA Kit
• Hydrogel Formulation Development