高分期刊重磅成果：靶向关键分子解析疾病深层作用机制，实现机制研究全新突破

Fig. 1.
Synthesis and characterization of composite hydrogels. (A) Schematic illustration of the hydrogel synthesis process. (B) SEM images of composite hydrogels. (C) Use 1 H NMR spectra to test CS-CAL. (D) Use of the UV–vis test CS-CAL and CAL. (E) FTIR spectra. Black arrows indicate specific stretching vibrations and bending vibrations. (F) 2DCOS spectra of amide bands in GelMA samples and GelMA/chitosan/polyphenol/PEG composite samples. (G) Amplitude sweep of hydrogels with shear strain ranging from 0.1 % to 1000 %. (H) Viscosity of the hydrogels plotted against the shear rate. ???P < 0.001.

Fig. 2.
Biocompatibility and functional characterization of the composite hydrogel. (A) Cytoskeletal staining of CPCs. (B) Live/dead staining results of CPCs cultured in different hydrogels. (C) Degradation profile of the composite hydrogel. (D–E) Release profiles of SDF-1α and EGTA. (F–G) Cell viability of hydrogels in CPCs and RAW264.7 cells after gelation at different time points. N.S., not significant.

Fig. 3.
The effect of composite hydrogels on ROS scavenging in CPCs. (A–B) H2DCFH- DA, a common ROS probe, was used to evaluate the antioxidant capability of CS-CAL@GS and CSE-CAL@GS. (C–D) Expression of antioxidant enzyme genes in CPCs evaluated by qRT-PCR. (E–F) Intracellular ROS levels detected by H2DCFH-DA staining and measured via FCM. (G–H) Fluorescence microscope images of JC-1 assay demonstrating activated (labeled red) and abnormal (labeled green) mitochondrial membrane potential. (I–K) Effects of CSE-CAL@GS on IL-6, TNF-α, and COX-2 mRNA levels in chondrocytes as determined by qRT-PCR. N.S., not significant; ?P < 0.05, ??P < 0.01, and ???P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4.
Effects of composite hydrogels on ROS levels and mitochondrial function in RAW264.7 cells. (A–B) Fluo-4 AM staining was used to visualize intracellular Ca2+ in LPS + Ca2+-induced M1 RAW264.7 macrophages. Untreated RAW264.7 cells served as the normal control. Semiquantitative fluorescence intensity analysis was performed using ImageJ. (C–D) The changes of Ca2+ concentration in M1 macrophages induced by LPS + Ca2+ were detected by flow cytometry. (E–F) H2DCFH-DA, a common ROS probe, was used to evaluate the ROS scavenging ability of CS-CAL@GS and CSE-CAL@GS. (G–H) Expression of antioxidant enzyme genes (Sod1 and Sod2) in RAW264.7 cells was evaluated by qRT-PCR. (I–J) Intracellular ROS levels were measured using H2DCFH-DA staining and flow cytometry. (K–L) Fluorescence microscope images of JC-1 assay demonstrating activated (labeled red) and abnormal (labeled green) mitochondrial membrane potential. ?P < 0.05, ??P < 0.01, and ???P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5.
Identification of macrophage polarization by immunofluorescence and flow cytometry. (A) Immunofluorescence staining of CD86+ (M1) macrophages in RAW264.7 cells. (B) Immunofluorescence staining of CD206+ (M2) macrophages in RAW264.7 cells. (C–D) Flow cytometric analysis of CD86+ (M1) and CD206+ (M2) macrophages.

Fig. 6.
Composite hydrogel reduces mitochondrial calcium overload and improves mitochondrial function in CPCs. (A–B) Western blot analysis of p-Drp1 expression. (C) Representative fluorescent images of CPCs stained with MitoTracker Green (mitochondria, green) and Rhod-2 AM (mitochondrial calcium, red). (D) SA-β-gal staining of CPCs from the four experimental groups. (E) Morphology of mitochondria detected by transmission electron microscopy. ?P < 0.05 and ???P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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