Dual-functional probiotic hydrogel with puerarin integration for microbiota-neuroimmune regulation in antibiotic-free periodontitis therapy

Abstract

Periodontitis is an inflammatory disease caused by the imbalance of the periodontal microbial ecosystem. Traditional treatment methods not only kill pathogenic bacteria but also inhibit the growth of beneficial bacteria, thereby disrupting the balance of the oral microbial ecosystem. In this study, chitosan, hyaluronic acid, and puerarin were coated on live Lactobacillus rhamnosus to form probiotic-based PSCLR nanoparticles. Subsequently, these nanoparticles were encapsulated in hydrogel microspheres through emulsion polymerization and immobilized in the hydrogel network via the covalent cross-linking of methacrylate-modified hyaluronic acid. This hydrogel restores the balance of oral microbiota by reducing pathogenic bacteria and promoting probiotic diversity, which helps maintain ecological stability and minimize risks associated with microbial community dysbiosis. In addition, puerarin in the gel achieves analgesic and neuromodulatory effects by specifically antagonizing the P2X₃ receptor, which affects pain transmission. This study provides a potential strategy for the antibiotic-free treatment of periodontitis and the neuropathic pain associated with it.

Keywords: Antibacterial; Hydrogel; Periodontitis; Probiotics; Puerarin.

Graphical abstract

Scheme 1

(A) Preparation of a puerarin-modified probiotic hydrogel; (B) Schematic diagram of flora regulation and neuroregulation mechanisms in periodontitis.

Fig. 1

Synthesis and characterisation of PSCLR. (A) SEM image of puerarin nanoparticles. (B) TEM images of puerarin nanoparticles. (C) High performance liquid chromatography of puerarin nanoparticles. (D) Schematic diagram of the synthesis of SCLR and PSCLR. SEM images of (E) LR, (F) SCLR, and (G) PSCLR. (H) Zeta potential values for different samples (LR, SCLR, and PSCLR) (n = 3 bio-independent samples). (I) FTIR diagrams of different samples (PNs, PSCLR, PSCLR-GHM and PSCLR-HA Gel).

Fig. 2

Synthesis and characterization of PSCLR-HA Gel. SEM images of (A) HAMA, (B) PSCLR-GHM, and (C) PSCLR-HA Gel. After culturing for 24 h in a specific MRS medium, we captured the (D) fluorescence field image and (E) three-dimensional image of PSCLR-GHM, as well as the (F) fluorescence field image and (G) three-dimensional image of PSCLR-HA Gel using a laser confocal microscope. (H) Probiotic nanoparticles coated with microsphere hydrogels (MHA) or uncoated (LHA) were cultured in MRS Medium. The OD values of MHA and LHA at 600 nm were measured after culture in MRS Medium. (I) Photos of injectable PSCLR-HA Gel. (J) Anti-plaque model. Physical tooth map and SEM image of bacteria in Sham, Control and PSCLR-HA Gel groups. (K) Bacterial live-dead staining of the biofilms in the control group and the PSCLR group.

Fig. 3

(A) Pictures of spread plates after incubating PSCLR at different concentrations with S. aureus for 24 h. (B) Pictures of spread plates after incubating PSCLR at different concentrations with E. coli for 24 h. (C) Pictures of spread plates after incubating PSCLR at different concentrations with F. nucleatum for 24 h. The CFU values after co-culturing PSCLR at different concentrations with (D) S. aureus, (E) E. coli, and (F) F. nucleatum. The in vitro antibacterial activities of the PSCLR-HA Gel against S. aureus, E. coli, and F. nucleatum. (H) Antimicrobial CFU diagram of live hydrogel against S. aureus, E. coli, and F. nucleatum in vitro.

Fig. 4

(A) Cell viability of NIH-3T3 cells measured by LDH after treatment with PSCLR, PSCLR-GHM, PSCLR-HA Gel and LDH release reagent (negative control) for 2, 4, and 24 h (n = 3 bioindependent samples). (B) Erythrocyte hemolysis test using PSCLR-HA Gel. (C) Antioxidant Test of PSCLR-HA Gel. (D) Fluorescence map of cells treated with PSCLR-HA Gel. FITC staining shows cytoskeletal actin in green and DAPI staining shows the nucleus in blue. Scale: 50 μm.

Fig. 5

(A) Flowchart of Animal Experiment. (B) Periodontal tissue sections stained with hematoxylin and eosin in the Sham group, Control group, PSCLR-HA Gel group and Periocline group. The average optical densities of (C) IL-10 and (D) TNF-α in periodontal tissues.

Fig. 6

After treatment, (A) the collagen deposition levels (blue areas) in periodontal tissue sections were observed by Masson staining, and (B) the expression of P2X₃ receptors in the trigeminal ganglion was detected by immunohistochemistry in the Sham group, Control group, PSCLR-HA Gel group, and Periocline group. Dark brown staining indicates positive expression of P2X₃ receptors.