Multifunctional thermo-sensitive hydrogel for modulating the microenvironment in Osteoarthritis by polarizing macrophages and scavenging RONS

Abstract

Osteoarthritis (OA) is a common degenerative joint disease that can lead to disability. Blocking the complex malignant feedback loop system dominated by oxidative stress and pro-inflammatory factors is the key to treating OA. Here, we develop a multifunctional composite thermo-sensitive hydrogel (HPP@Cu gel), which is utilized by Poloxamer 407 (P407) and hyaluronic acid (HA) mixture as the gel matrix, then physically mixed with copper nanodots (Cu NDs) and platelet-rich plasma (PRP). Cu NDs is a novel nano-scavenger of reactive oxygen and nitrogen species (RONS) with efficient free radical scavenging activity. HPP@Cu gel is injected into the articular cavity, where it form an in situ gel that slowly released Cu NDs, HA, and PRP, prolonging the duration of drug action. Our results indicate that HPP@Cu gel could efficiently remove RONS from inflammatory sites and promote repolarization of macrophages to an anti-inflammatory phenotype. The HPP@Cu gel therapy dramatically reduces cartilage degradation and inflammatory factor production in OA rats. This study provides a reliable reference for the application of injectable hydrogels in inflammatory diseases associated with oxidative stress.

Keywords: Copper nanodots; Macrophage polarization; Osteoarthritis; Reactive oxygen and nitrogen species; Thermo-sensitive hydrogels.

Fig. 1

Schematic illustration for the synthesis processes of HPP@Cu thermoresponsive hydrogel and treatment mechanism as articular microenvironment purifier for OA

Fig. 2

Physical characterizations and RONS scavenging activity of Cu NDs. a TEM image of Cu NDs. Scale bars = 50 μm. b Hydrodynamic diameter distribution of Cu NDs. c XRD pattern of Cu NDs. d FTIR spectra of Cu NDs. e UV-vis spectra of Cu NDs. f H₂O₂ scavenging activity of Cu NDs at various concentrations. g NO· scavenging activity of Cu NDs at various concentrations. h ·O₂^- scavenging activity of Cu NDs at various concentrations. i ·PTIO scavenging activity of Cu NDs at various concentrations. j NO· scavenging activity of Cu NDs at various concentrations. k ABTS·⁺ scavenging activity of Cu NDs at various concentrations. The inset is ABTS·⁺ test solution treated with various concentrations of Cu NDs. l ·DPPH scavenging activity of Cu NDs at various concentrations. The inset is ·DPPH solution treated with various concentrations of Cu NDs. In f–l, data represent means ± SD from three independent replicates

Fig. 3

a The viscosity profile of the P407 hydrogels at various concentrations (15%, 18%, 20%, 22%, 25%) altered with increasing temperature. b Variation of viscoelastic parameters (η*, G’, G’’) of 18% P407 at different temperatures. c The viscosity profile of the P407 hydrogels (18%) after adding HA at various concentrations (0.1%, 0.15%, 0.2%, 0.25%) altered with increasing temperature. d Variation of viscoelastic parameters (η*, G’, G’’) of 18% P407 added with 0.20% HA at different temperatures. e SEM of HP gel and HPP@Cu gel. Scale bars = 100 μm. f Images of HPP@Cu gel at 4 ℃ or 37 ℃. g Release profile of Cu NDs loaded by HPP@Cu gel. Data represent means ± SD from three independent replicates

Fig. 4

a Cytotoxicity of Cu NDs on L929 and chondrocyte. b Cytotoxicity of Cu NDs on activated RAW264.7 and inactivated RAW264.7. c Cell viability of L929 and chondrocyte with different treatments. d Cell viability of activated RAW264.7 and inactivated RAW264.7 with different treatments. e Fluorescence images of Calcein-AM/PI staining of chondrocyte after incubation with different concentrations of PRP in HPP@Cu gel. Scale bars = 100 μm. f Cell viabilities of chondrocyte under different treatment conditions. Data represent means ± SD, n = 5. **p < 0.01, ##p < 0.01, and &&p < 0.01. NS means not significant. g Quantification of the ratio of apoptotic and living cells by flow cytometry analysis according (h). h Flow cytometry analysis of apoptotic and necrotic cells under different treatment conditions. In a–d, data represent means ± SD, n = 5

Fig. 5

RONS scavenging capacity of HPP@Cu gel for RAW264.7 and chondrocyte. a Confocal fluorescence images of the levels of ROS of RAW264.7. b Confocal fluorescence images of the levels of RNS of RAW264.7. c Confocal fluorescence images of the levels of ROS of chondrocyte. d Confocal fluorescence images of the levels of RNS of chondrocyte. e–h Corresponding to a–d, the mean fluorescence intensity was quantified by ImageJ, respectively. In e–h, data represent means ± SD, n = 3. *p < 0.05, #p < 0.05, and &p < 0.05. **p < 0.01, ##p < 0.01, and &&p < 0.01. NS means not significant

Fig. 6

Effect of HPP@Cu gel on macrophage phenotype. a Immunofluorescence analysis of markers of M1 (iNOS, green) and M2 (CD206, red) on macrophages after different treatments. b Corresponding to a, the mean fluorescence intensity of iNOS was quantified by ImageJ. c Corresponding to a, the mean fluorescence intensity of CD206 was quantified by ImageJ. d Flow cytometry analysis of the percentage of M2 in the macrophage population. In b, c, data represent means ± SD, n = 3. **p < 0.01, ##p < 0.01, and &&p < 0.01. NS means not significant

Fig. 7

Histological analysis after treatment in vivo. a Schematic illustration of the establishment and treatment schedule of MIA-induced OA rats. b Pelletier score analysis according macroscopic appearance, and OARSI score analysis according H&E staining and Safranin O staining. Data represent means ± SD, n = 3. &p < 0.05. **p < 0.01, and ##p < 0.01. NS means not significant. c Macroscopic appearance of joints after treatment. d H&E staining and Safranin O staining of joints after treatment. Scale bars = 50 μm

Fig. 8

a Levels of ROS in joint tissue homogenate. b Levels of RNS in joint tissue homogenate. c Levels of IL-1β in joint tissue homogenates. d Levels of TNF-α in joint tissue homogenates. e Levels of IL-10 in joint tissue homogenates. f Immunofluorescence staining of iNOS (M1 marker, red) and CD206 (M2 marker, green) of synovial tissue. g Liver function indicators levels and kidney function indicators levels of serum after treatment. In a–e, and g, data represent means ± SD, n = 3. *p < 0.05, #p < 0.05. **p < 0.01, ##p < 0.01, and &&p < 0.01. NS means not significant