Regulation of rheumatoid arthritis microenvironment via a self-healing injectable hydrogel for improved inflammation elimination and bone repair

Abstract

The rheumatoid arthritis (RA) microenvironment is often followed by a vicious circle of high inflammation, endogenous gas levels imbalance, and poor treatment. To break the circle, we develop a dual-gas-mediated injectable hydrogel for modulating the immune microenvironment of RA and simultaneously releasing therapeutic drugs. The hydrogel (DNRS gel) could be broken down on-demand by consuming excessive nitric oxide (NO) and releasing therapeutic hydrogen sulfide (H₂S), resulting in endogenous gas restoration, inflammation alleviation, and macrophage polarization to M2 type. Additionally, the hydrogel could suppress osteoclastogenesis and enhance osteogenesis. Furthermore, the intra-articularly injected hydrogel with methotrexate (MTX/DNRS gel) significantly alleviated inflammation and clinical symptoms and promoted the repair of bone erosion in the collagen-induced arthritis rat model. As a result, in vivo results demonstrated that MTX/DNRS gel restored the microenvironment and improved the therapeutic effect of MTX. This study provides a novel understanding of developing versatile smart delivery platforms for RA treatment.

Keywords: Hydrogels; Hydrogen sulfide; Microenvironment; Nitric oxide; Rheumatoid arthritis.

Graphical abstract

Scheme 1

Schematic illustration of the fabrication of self-healing injectable hydrogel and the mechanism of bone protection by regulating the microenvironment of RA. DNRS gel depletes excessive NO and releases H₂S in the presence of cysteine, and sustained release MTX.

Fig. 1

Pathological features of rats in CIA and normal groups. a, b) immunofluorescence analyses of CD86 macrophages (green), immunohistochemical analyses, iNOS expression, and TRAP staining of osteoclast number in joints, micro-CT images of ankles respectively. c, d) Quantitative micro-CT analysis of BMD and BS/BV (n = 3). **P < 0.01, ***P < 0.001.

Fig. 2

Characterization of DNRS gel. a) The polymerization mechanism and photograph of DNRS gel. b) SEM images of DNRS gel. c) Step-strain oscillatory rheology of the self-healing DNRS gel (Step-strain from ε = 0.5%–200%, angular frequency = 10 rad s⁻¹, 25 °C). d) Time-sweep rheological properties for DNRS gel (ε = 0.5%, angular frequency = 10 rad s⁻¹, 25 °C). e) Schematic illustration of the self-healing gel, hydrophobic cross-linkers in gels can be reconnected under physical strain. f) The NO scavenging and g) H₂S releasing of DNRS gel. h) The DNRS gel images after 4 h incubation in PBS, NO solution (40 μM), and cysteine solution (100 μM). i) Relative swelling and j) degradation rates of DNRS hydrogels in PBS, NO solution (40 μM), and cysteine solution (100 μM) at different times, respectively. k) The working principle of DNRS gel. It depletes NO via the transformation of o-phenylenediamine into carboxyl and releases H₂S by the cysteine-triggered H₂S donor. l) The SEM images of the degraded DNRS gel. MTX red-loaded DNRS gel and Ctrl gel release profile after incubation with m) NO solution, n) cysteine, and o) NO and cysteine.

Fig. 3

In vitro anti-inflammation effects of DNRS gel. a) RAW264.7 viability was determined using CCK-8 assay for 24h treatment with DNRS gel and Ctrl gel at various concentrations (n = 4). b) Live/dead staining of macrophages and osteoblasts treated with Ctrl gel and DNRS gel. c) Intracellular NO level and H₂S level in LPS-activated macrophages treated with Ctrl gel and DNRS gel (n = 4). The corresponding fluorescence intensities of d) H₂S and e) NO in macrophages after different treatments. f-h) TNF-α, IL-6, and anti- IL-10 levels of different groups (n = 5). i) The CD86 and CD206 expression levels of macrophages after different treatment by the flow cytometry analysis. j) Representative fluorescent images of CD86 and CD206 in macrophages after different treatments. Scale bar: 20 μm. k) GO enrichment and l) KEGG enrichment analyses within LPS and DNRS gel groups. m) The potential mechanism of anti-inflammation macrophage and macrophage polarization by depleting NO and releasing H₂S. n.s. represents not significant, *P < 0.05, **P < 0.01, ****P < 0.0001, comparison between # and other groups.

Fig. 4

DNRS gel improved the osteogenic potential under the inflammatory environment. a) The collection of conditional medium and osteoblasts processing method. Representative images of b) ALP staining and d) alizarin red staining of osteoblasts after treatment with different conditional media. Scale bar: 500 μm. Quantification analysis of c) ALP activity and d) mineralization. f-h) The levels of Col I, OCN, and ALP were measured by ELISA. i) CLSM images of multinucleated cells after different treatments for 3 days (green: F-actin; blue: nuclei). Scale bar: 20 μm. j) The production of TRAP of RAW264.7 cells after different treatments for 3 days. n = 4, n.s. represents not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5

In vivo therapeutic effects of DNRS gel. a) Experimental timeline of gel treatment. b) Clinical score and c) body weight of CIA rats for different groups. d) Thermographic images and e) temperature of inflammatory joints after various treatments on different days. f) H&E staining of synovium. g) H&E and Masson staining of ankle joints. Scale bars: 100 μm. n = 6, n.s. represents not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 6

In vivo anti-inflammatory effects of MTX/DNRS gel. a) The immunofluorescence staining of CD206 and iNOS in the joints of different groups. Scale bar: 50 μm. b-g) The secretion of TNF-α, IL-6, and IL-10 in the serum and tissues of different groups. h-i) NO and H₂S concentration in the joint synovial fluid at day 28. n = 4, n.s. represents not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 comparison between # and other groups.

Fig. 7

Bone protection of MTX/DNRS gel in joints of CIA rats. a) ALP, Col I, and OCN expression levels and TRAP-stained osteoclasts in the joints in different groups. Scale bar: 100 μm. b-e) Statistics of ALP, Col I, OCN changes, and numbers of osteoclasts in the joints. f-g) BMD and BS/BV of RA rats received different treatments at day 28. h) Representative photographs and micro-CT images of RA rats received different treatments on day 28. n = 3, n.s. represents not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.