Multifunctional injectable hydrogel incorporating EGCG-Cu complexes for synergistic antibacterial, immunomodulatory, and osteogenic therapy in periodontitis

Abstract

Periodontitis is a chronic inflammatory disease characterized by oxidative stress, immune system imbalance, and the progressive destruction of periodontal tissues. Traditional treatment approaches are limited by the incomplete eradication of pathogens, the risk of antibiotic resistance, inadequate control of inflammation and oxidative stress, and restricted tissue regeneration capacity. Therefore, this study proposes an injectable multifunctional Laponite/gelatin hydrogel loaded with epigallocatechin gallate (EGCG)-copper ion (Cu²⁺) complexes as a localized therapy for periodontitis. EGCG exhibits antioxidant, anti-inflammatory, and antimicrobial properties; however, its clinical application is hindered by poor stability and bioavailability. Cu²⁺ coordination enhances EGCG stability and antioxidant capacity while improving its antimicrobial efficacy. Experimental results demonstrate that the Laponite/gelatin hydrogel is adaptable for the localized delivery of EGCG-Cu, with bioactive metal ions such as Li⁺, Mg²⁺, and Si⁴⁺ contained in Laponite, which promote osteogenesis and periodontal tissue regeneration. In vitro and in vivo studies confirm that this hydrogel exhibits excellent biocompatibility, effectively inhibits Porphyromonas gingivalis, suppresses M1 polarization while promoting M2 polarization, and facilitates periodontal tissue repair. Therefore, this study provides promising insights into a localized therapeutic strategy for periodontitis.

Keywords: Alveolar bone regeneration; Copper ions (Cu2+); Epigallocatechin gallate (EGCG); Hydrogel; Periodontitis.

Graphical abstract

Fig. 1

Schematic illustration of the fabrication and application of Lap-Gel/E-Cu hydrogel for periodontitis treatment. (A) Schematic diagram of the synthesis of EGCG-Cu complex. (B) Schematic diagram of the synthesis of Lap-Gel/E-Cu hydrogel. (C) Schematic representation of the antibacterial, ROS-scavenging, immunomodulatory and osteogenesis-promoting functions of Lap-Gel/E-Cu hydrogel in the treatment of periodontitis.

Fig. 2

Morphological and elemental characterization of EGCG-Cu. (A) TEM images of EGCG-Cu. (B) The elemental mapping images of EGCG-Cu complexing compound. (C) XPS spectrogram of EGCG. (D) XPS spectrogram of EGCG-Cu. (E) O1s XPS spectra of EGCG. (F) O1s XPS spectra of EGCG-Cu. (G) UV–vis absorption spectra of EGCG and EGCG-Cu. (H) FTIR spectra of EGCG and EGCG-Cu.

Fig. 3

Characterization of the Lap-Gel/H-E-Cu hydrogels. (A) The SEM images of the Lap-Gel, Lap-Gel/L-E-Cu, Lap-Gel/M-E-Cu, and Lap-Gel/H-E-Cu hydrogels. (B) Elemental mapping of Lap-Gel/H-E-Cu hydrogel. (C) Different shapes of hydrogels. (D) and (G) self-healing property of hydrogels over time (E) Adhesion to premolar. (F) Injectability of hydrogels through the syringe. (H) Schematic representation of testing hydrogel injection force with mechanical tester. (I) Extrusion force vs time was recorded, and the arrow indicates the maximum required force to injecting the hydrogel. (J) Injection force of Lap-Gel was determined in different velocities of flow. (K) Injection force of Lap-Gel, Lap-Gel/L-E-Cu, Lap-Gel/M-E-Cu, and Lap-Gel/H-E-Cu. (L) Shear stress versus shear rate for the Lap-Gel/H-E-Cu hydrogels. (M) Viscosity of the Lap-Gel/H-E-Cu hydrogels as a function of shear rate. (N) The G′ and G″ values were recorded when the Lap-Gel/H-E-Cu hydrogels were subjected to cyclic strain changes between 0.1 % and 100 % strain. (O) FTIR spectra of Lap-Gel, Lap-Gel/L-E-Cu, Lap-Gel/M-E-Cu, and Lap-Gel/H-E-Cu hydrogels.

Fig. 4

In vitro antibacterial properties of the hydrogels. (A) Schematic diagram of antibacterial experiment steps. (B) Images of S. aureus and P. gingivalis bacterial colonies treated with different hydrogel groups. (C) Live/dead vitality image of S. aureus and P. gingivalis treated with various hydrogels. (D) Quantitative analysis of the antimicrobial rates against S. aureus. (E) Quantitative analysis of the antimicrobial rates against P. gingivalis. (F) Live/dead bacterial rate of S. aureus treated with various hydrogels. (G) Live/dead bacterial rate of P. gingivalis treated with various hydrogels.

Fig. 5

Hemocompatibility and cytocompatibility testing of hydrogels. (A) Blood image after co-culture with the hydrogel and erythrocyte morphology under the microscope. (B) Hemolysis rate of the hydrogels. Live/dead fluorescent staining of (C) RAW264.7 cells and (D) BMSCs. Cell viability of (E) RAW264.7 cells and (F) BMSCs at days 1, 3, and 5.

Fig. 6

Antioxidant properties of hydrogels. (A) Absorbance curves changes of staining appearance in different groups of hydrogel extractions added to DPPH solution. Absorbance curves (B) and scavenging ratios (C) of hydrogels with varying concentrations of nanosheets of DPPH assays. (D) DCFH-DA staining and (E) fluorescent quantification results. (F) Live/dead staining and (G) CCK-8 assay under oxidative stress conditions.

Fig. 7

Macrophage polarization modulation by hydrogels. IF images showing hydrogels modulating macrophage polarization for (A) M1-type macrophage markers (iNOS) and (B) M2-type macrophages markers (CD206). (C) Fluorescence intensity of iNOS. (D) Fluorescence intensity of CD206. (E) Schematic diagram showing the effect of hydrogels on macrophage polarization.

Fig. 8

In vitro assessment of the osteogenic differentiation potential of hydrogels. (A) Schematic diagram outlining the experimental procedure for ALP, ARS, and IF staining to evaluate BMSC osteogenic differentiation. (B) ALP staining (day 4) and ARS (day 21) of BMSCs respectively. (D) IF staining images of OCN. (E) IF staining images of RUNX2.

Fig. 9

Bone regeneration assessments in vivo. (A) The schematic diagram of hydrogel treatment of periodontitis in SD rats. (B) The modeling of periodontitis in SD rats was completed. (C) The maxillae were analyzed by Micro-CT. (D) CEJ-ABC distance measured from micro-CT images. (E) 3D reconstructed and 2D images after 4 weeks of implantation. (F) BMP2 and OCN immunohistochemical image. (Black arrows indicate high expression areas).

Fig. 10

In vivo assessment of periodontal inflammation pathology in hydrogel-treated rats. (A) H&E staining of maxillae from the control, periodontitis, and two hydrogel treatment groups. (B) Masson's trichrome staining of maxillae across the control, periodontitis, and hydrogel treatment groups. (C) IL-10 and TNF-α stained images (Black arrows indicate high expression areas). AB: alveolar bone.