Dynamic hydrogel-metal-organic framework system promotes bone regeneration in periodontitis through controlled drug delivery

Abstract

Periodontitis is a prevalent chronic inflammatory disease, which leads to gradual degradation of alveolar bone. The challenges persist in achieving effective alveolar bone repair due to the unique bacterial microenvironment's impact on immune responses. This study explores a novel approach utilizing Metal-Organic Frameworks (MOFs) (comprising magnesium and gallic acid) for promoting bone regeneration in periodontitis, which focuses on the physiological roles of magnesium ions in bone repair and gallic acid's antioxidant and immunomodulatory properties. However, the dynamic oral environment and irregular periodontal pockets pose challenges for sustained drug delivery. A smart responsive hydrogel system, integrating Carboxymethyl Chitosan (CMCS), Dextran (DEX) and 4-formylphenylboronic acid (4-FPBA) was designed to address this problem. The injectable self-healing hydrogel forms a dual-crosslinked network, incorporating the MOF and rendering its on-demand release sensitive to reactive oxygen species (ROS) levels and pH levels of periodontitis. We seek to analyze the hydrogel's synergistic effects with MOFs in antibacterial functions, immunomodulation and promotion of bone regeneration in periodontitis. In vivo and in vitro experiment validated the system's efficacy in inhibiting inflammation-related genes and proteins expression to foster periodontal bone regeneration. This dynamic hydrogel system with MOFs, shows promise as a potential therapeutic avenue for addressing the challenges in bone regeneration in periodontitis.

Keywords: Bone regeneration; Drug delivery; Hydrogel; Metal-organic frameworks; Periodontitis.

Fig. 1

The schematic illustration of the preparation, application and bone regeneration promotion mechanism of CSBDX@MOF

Fig. 2

The characterization of CSBDX and Mg-GA. A The FT-IR spectrum of CSBDX and each component. B Self-healing property of CSBDX. Two pieces of self-healed hydrogel can support its own weight and formation of dynamic Schiff base and boronic ester bond and self-healing interface was observed with microscope and continuous step-strain measurement. C The injectability of CSBDX. D The morphology and EDS mapping images of Mg-GA (bar = 2 μm). E The XRD spectrum of Mg-GA. F The structure schematic diagram of Mg-GA. G The XPS spectrum of Mg-GA and peak-differentiating and imitating of Mg element

Fig. 3

The characterization of CSBDX@MOF. A The morphology, EDS mapping images and energy spectrum of CSBDX@5MOF. The white indicates Mg-GA particles (bar = 50 μm). B The cross-sectional morphology and pore diameter histogram of CSBDX@MOF (bar = 100 μm, n = 6). C The visualizations of surface morphology and Sa (the arithmetic average height deviation from a mean plane) values of different surfaces of CSBDX@MOF (n = 6). D The water contact angles of CSBDX@MOF (n = 3). E The representative tensile stress–strain curves, fracture strain and largest tensile strength of CSBDX@MOF (n = 6). F The representative compressive stress–strain curves and compressive modulus of CSBDX@MOF (n = 6). G The cyclic compressive stress–strain curves of CSBDX@MOF (n = 6). The swelling profile H and degradation profile I of CSBDX@MOF (n = 4). J The Mg-GA releasing profile of CSBDX@10MOF in different condition. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: no significance

Fig. 4

Antibacterial effect. A The schematic illustration of antibacterial effect of hydrogel system on A. a. and P. gingivalis. B Representative images of Live/dead bacteria staining of A. a. and P. gingivalis after co-culture with hydrogel (bar = 100 μm). C Representative images of colony formation assay and bacterial survival histogram D of A. a. and P. gingivalis after co-culture with hydrogel and corresponding statistical analysis of bacterial survival (n = 4). E Representative SEM images of A. a. and P. gingivalis after co-culture with hydrogel (bar = 2 μm). Yellow pseudo-color indicates the stacked and crumpled bacteria. *: P < 0.05, ***: P < 0.001, ****: P < 0.0001, ns no significance

Fig. 5

The biocompatibility of CSBDX@MOF. A Hemolysis ratio of rabbit erythrocytes incubated with hydrogels (n = 4). The RAW264.7s B and MC3T3 cells C proliferation quantitatively analyzed by CCK-8 after treated with hydrogel extracts for 1, 3, and 5 days (n = 4). Representative live/dead staining of RAW264.7s E and MC3T3 cells E cultured with hydrogel extracts for 5 days (bar = 200 μm). The RAW264.7s F and MC3T3s G morphology after cultured with hydrogel extracts for 5 days (bar = 50 μm). *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001

Fig. 6

The antioxidant and immunomodulatory properties of CSBDX@MOF. The quantitative analysis of DPPH A and ABTS B radical scavenging efficiency of hydrogel. C Representative flow cytometry data and mean fluorescence intensity of DCF in RAW264.7 treated with 300 μM H2O2 with hydrogel extracts. D Representative DCFH-DA staining images of in RAW264.7 treated with 300 Μm H2O2 (bar = 200 μm). Relative gene (pro-inflammatory: iNOS, COX-2 and IL-6. Anti-inflammatory: TGF-β3, IL-10 and CD206) expressions E and macrophage polarization-related protein (iNOS and CD206) expressions F in RAW264.7 incubated with hydrogel extracts with stimulation of 1 μg/mL P.g.-LPS for 24 h (bar = 50 μm). G Flow cytometry of macrophage polarization surface markers (CD86 and CD206). *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001

Fig. 7

The osteogenic effect of CSBDX@MOF. Relative gene expression of RUNX2, ALP, COL1, OPN, OCN and OPG in MC3T3 cells incubated with macrophage conditioned medium on 7th day A and 14th day B (n = 4). C ALP staining of MC3T3 cells incubated with macrophage conditioned medium on 7th day (bar = 500 μm). D ARS staining of MC3T3 cells incubated with macrophage conditioned medium on 21st day (bar = 500 μm, n = 4). The representative immunofluorescence images of OCN (E) and RUNX2 (F) of MC3T3 cells incubated with macrophage conditioned medium on 7th and 14th day (bar = 50 μm). G The statistical analysis of area of ALP and ARS staining. H The statistical analysis of mean fluorescence intensity (MFI) of OCN and RUNX2 (n = 4). *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns no significance

Fig. 8

Evaluation of alveolar bone regeneration using micro-CT. A The schematic diagram of animal experiment. Representative micro-CT three-dimensional reconstruction and section images and CEJ-ABC distance, BV/TV quantitative analysis on the 7th day B and 28th day C (n = 6). *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns no significance

Fig. 9

Histological evaluation of immunomodulatory, collagen deposit and alveolar bone regeneration of hydrogels. Representative H&E staining images on the 7th day A (bar = 1 mm upper, bar = 100 μm lower). Representative immunohistochemical staining images of iNOS B and CD206 C on the 7th day and the statistical analysis of positive cells of iNOS and CD206 per HPF (bar = 100 μm, n = 6). Representative Masson’s trichrome staining images D (bar = 1 mm upper, bar = 100 μm lower). Representative immunohistochemical staining images of COL1 E and OCN F on the 28th day and the statistical analysis of positive cells of COL 1 and OCN per HPF (bar = 100 μm, n = 6). *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns no significance