Lacc1-engineered extracellular vesicles reprogram mitochondrial metabolism to alleviate inflammation and cartilage degeneration in TMJ osteoarthritis

Abstract

Temporomandibular joint osteoarthritis (TMJOA) is a multifaceted degenerative disease characterized by progressive cartilage degradation, chronic pain, and functional limitations of the TMJ, significantly affecting patients' quality of life. Although metabolic homeostasis in chondrocytes is crucial for cartilage health, the mechanisms underlying metabolic dysregulation in TMJOA remain poorly characterized. This study aimed to investigate the metabolic imbalance in TMJOA cartilage and explore novel therapeutic strategies targeting metabolic reprogramming. RNA sequencing revealed a significant imbalance between glycolysis and oxidative phosphorylation (OXPHOS) in TMJOA cartilage, with a marked shift toward glycolysis, which is associated with inflammation and cartilage degradation. To counteract this imbalance, Laccase domain-containing 1 (Lacc1), a metabolic regulator involved in both inflammation and metabolic homeostasis, was selected for investigation, as its role in chondrocytes had not been explored. We engineered macrophage-derived extracellular vesicles (EVs) to overexpress Lacc1 (OE-EVs), aiming to restore metabolic balance and modulate inflammation in chondrocytes. In vitro, OE-EVs significantly reduced IL-1β-induced inflammation, inhibited glycolysis by decreasing key glycolytic enzymes, improved mitochondrial function by decreasing mitochondrial superoxide levels, and the restoration of normal mitochondrial structure. In vivo, micro-computed tomography (Micro-CT) and histological analyses demonstrated that OE-EVs effectively alleviated inflammation and promoted cartilage repair, as indicated by a 1.55-fold increase in toluidine blue-stained cartilage area compared to the TMJOA group, reflecting improved cartilage matrix integrity and proteoglycan retention. These findings highlight the therapeutic potential of Lacc1-engineered EVs to target mitochondrial metabolism, reestablish metabolic homeostasis, and reduce inflammation in TMJOA, offering a novel and promising strategy for improving clinical outcomes in TMJOA patients.

Keywords: Chondrocytes; Engineered extracellular vesicles; Laccase domain-containing 1 (Lacc1); Mitochondrial metabolism; Temporomandibular joint osteoarthritis (TMJOA).

Fig. 1

The application of Lacc1-engineered EVs in TMJOA chondrocytes. (A) The construction of Lacc1-engineered EVs. (B) Lacc1-engineered EVs alleviated TMJOA inflammation via modulating mitochondrial metabolism in chondrocytes. (By Figdraw.)

Fig. 2

Metabolic Dysregulation in TMJOA Cartilage. (A) Volcano plot of differentially expressed genes in TMJOA cartilage. (B) KEGG pathway enrichment analysis of metabolism-related signaling pathways. (C&D) qRT-PCR analysis of glycolytic enzymes, Lacc1, and inflammatory cytokines in TMJOA cartilage. (E&F) Immunofluorescence staining of Lacc1 and PKM2 in TMJOA cartilage. (n = 3, *p < 0.05, **p < 0.01, and ***p < 0.001)

Fig. 3

OE-EVs attenuate IL-1β-induced inflammation in chondrocytes. (A) TEM and NTA characterization of NC-EVs and OE-EVs. (B) PKH26-labeled EVs internalized by chondrocytes. (C) Western blot analysis of exosomal markers and Lacc1 in NC-EVs and OE-EVs. (D) qRT-PCR of inflammatory and cartilage matrix markers. (E&G) Immunofluorescence staining of Aggrecan and MMP13. (F) IL-1β levels measured by ELISA. (H) Western blot of inflammation- and ECM-related markers. (n = 3, *p < 0.05, **p < 0.01, and ***p < 0.001)

Fig. 4

OE-EVs reprogram metabolism in chondrocytes. (A&B) qRT-PCR and Western blot analysis of glycolytic enzyme expression. (C&D) ATP production and lactate levels in chondrocytes. (E) JC-1 staining of mitochondrial membrane potential (red: JC-1 aggregates, green: JC-1 monomers). (F) Mitochondrial ROS analysis using MitoSOX and Mitotracker staining. (G) TEM images of mitochondria in different groups. (n = 3, *p < 0.05, **p < 0.01, and ***p < 0.001)

Fig. 5

Transcriptomic and signaling pathway analysis of IL-1β- and OE-EV-treated chondrocytes. (A) Heatmap of differentially expressed genes. (B&C) KEGG and GO enrichment analysis. (D) GSEA analysis of key pathways. (E&F) Western blot and semi-quantification of JAK-STAT signaling. (n = 3, *p < 0.05 and **p < 0.01)

Fig. 6

OE-EVs promote cartilage repair and suppress inflammation in TMJOA in vivo. (A) Schematic of OE-EV treatment in a TMJ-OA mouse model. (B) In vivo fluorescence imaging of TMJ-injected OE-EVs and NC-EVs. (C&D) Micro-CT images and bone structural parameters. (E-G) Histological staining (H&E, Safranin O-Fast Green, Toluidine Blue). (n = 6, *p < 0.05, and ***p < 0.001)

Fig. 7

Histological and immunohistochemical analysis of TMJ condyle tissue. (A&B) Immunohistochemical staining for Collagen II and IL-1β. (C-E) Immunofluorescence staining of Aggrecan, MMP13, and MMP3. (F-H) Quantification of fluorescence intensity. (I) OARSI scoring for cartilage degradation. (n = 3, **p < 0.01, and ***p < 0.001)