Dysregulation of Glycolysis and OXPHOS homeostasis in TMJOA cartilage
To discover gene expression alterations within the condyles of TMJOA mice, condyle tissues from each regular and TMJOA mice have been collected for transcriptomic evaluation. As Fig. 2A depicted, sequencing outcomes demonstrated a big upregulation of glycolysis-related genes, together with HK3 (Hexokinase 3) and Pklr (Pyruvate kinase). In distinction, key genes associated to oxidative phosphorylation (OXPHOS) and TCA cycle, together with Cs (Citrate synthase), Mdh2 (Malate dehydrogenase), and Sdhc (Succinate dehydrogenase), have been markedly downregulation.
Hexokinase (HK), together with HK1, HK2, HK3, HK4, and HKDC1, play vital roles in glucose metabolism, catalyzing the preliminary step of glucose utilization [25]. The upregulation of HKs suggests enhanced glycolytic exercise. Citrate synthase (Cs) initiates the tricarboxylic acid (TCA) cycle by catalyzing the condensation of oxaloacetate and acetyl-CoA [26]. Furthermore, Mdh2, a vital enzyme within the TCA cycle, contributes to power manufacturing and stabilizes hypoxia-inducible issue 1 alpha (HIF-1α), linking metabolism to mobile responses to hypoxia [27]. Sdhc, serves as a pivotal enzyme bridging the TCA cycle and OXPHOS pathway, its downregulation additional signifies disrupted power metabolism in TMJOA cartilage [28].
Moreover, the expression of structural cartilage matrix genes, Col2a1 (collagen II) and acan (aggrecan) was considerably decreased, whereas IL-1β and matrix metalloproteinases 10 (MMP10) ranges have been markedly elevated within the TMJOA condylar cartilage. Kyoto Encyclopedia of Genes and Genomes (KEGG) evaluation indicated that differentially expressed genes have been primarily enriched in pathways associated to “Oxidative phosphorylation”, “Citrate cycle (TCA cycle)”, “Glycolysis/Gluconeogenesis” and others (Fig. 2B). These alterations point out a metabolic shift in the direction of enhanced glycolytic exercise in TMJOA condylar cartilage, contrasting with a lower in TCA cycle and OXPHOS performance in comparison with regular cartilage.
To validate the expression of key metabolic enzymes, qRT-PCR was carried out, revealing important upregulation of glycolysis-associated genes, together with HK1, HK2, Lactate dehydrogenase (LDHA), and Pyruvate kinase (PKM2) in OA cartilage (Fig. 2C). The expression ranges of Lacc1 and pro-inflammatory cytokines have been additionally assessed. As proven in Fig. 2D, pro-inflammatory markers comparable to IL-1β, matrix metalloproteinases (together with MMP9, MMP13) and tumor necrosis factor-α (TNF-α) have been markedly elevated in OA cartilage. Earlier researches indicated that IL-1β performed a vital function in OA by selling the upregulation of matrix metalloproteinases (MMPs), which contributed to the irreversible breakdown of extracellular matrix (ECM) and development of OA [1, 29]. In distinction, each qRT-PCR outcomes (Fig. 2D) and immunofluorescence staining for Lacc1 (Fig. 2E) demonstrated a big downregulation of Lacc1 in TMJOA cartilage, suggesting its potential anti-inflammatory function and its relationship with glycolysis in chondrocytes.
Moreover, Fig. 2F illustrated a pronounced expression of PKM2, a key glycolytic enzyme, in TMJOA group. Earlier research have highlighted that the metabolism reprogramming performed an important function in knee OA, with infected chondrocytes shifting their metabolism in the direction of glycolysis, leading to LDHA accumulation and elevated reactive oxygen species (ROS) era [29]. Enzymes such like pyruvate kinase (PKM), lactate dehydrogenase (LDHA) and hexokinase (HK) are integral to regulating glucose metabolism, and IL-1β induction considerably elevated the expression of these genes in chondrocytes [29, 30]. Collectively, our findings point out an imbalance between glycolysis and OXPHOS in TMJOA cartilage, with Lacc1 probably performing as a metabolic hyperlink within the pathogenesis of TMJOA.
Metabolic Dysregulation in TMJOA Cartilage. (A) Volcano plot of differentially expressed genes in TMJOA cartilage. (B) KEGG pathway enrichment evaluation of metabolism-related signaling pathways. (C&D) qRT-PCR evaluation 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)
Validation of the operate of Lacc1 in RAW264.7 macrophages
To elucidate the function of Lacc1 in RAW264.7 macrophages, we carried out each siRNA-mediated knockdown and Lacc1 overexpression experiments. Initially, qRT-PCR was employed to establish the simplest siRNA for Lacc1 knockdown (Fig. S1A). The optimum siRNA was chosen for subsequent evaluation to guage its impact on macrophage polarization upon LPS therapy. As Fig. S1B depicted, siLacc1 therapy elevated the expression of pro-inflammatory cytokines together with TNF-α, IL-1β and inducible nitric oxide synthase (iNOS), whereas considerably decreasing the expression degree of Arginase-1 (Arg-1), a marker related to anti-inflammatory M2 macrophages [31, 32], suggesting a shift towards a pro-inflammatory phenotype in siLacc1-treated macrophages. Subsequently, the expression of glycolysis associated genes have been additionally assessed by qRT-PCR. Fig. S1C illustrated that siLacc1 markedly elevated the expression of aconitate decarboxylase 1 (Acod1), PKM2, phosphofructokinase (PFKp) and HK1, which served necessary roles within the technique of glycolysis [25, 33]. Moreover, ATP degree measured in Fig. S1D indicated that siLacc1 led to decrease ATP manufacturing, no matter LPS stimulation, in comparison with the siNC group.
Subsequently, Lacc1 overexpression plasmids have been transfected into RAW264.7 macrophages to evaluate the results of Lacc1 on irritation and metabolism. The improved expression of Lacc1 within the transfected cells, in comparison with the detrimental management (NC) group, was confirmed by way of fluorescence microscopy, quantitative real-time PCR (qRT-PCR), and western blot analyses (Fig. S2A-S2C). Then qRT-PCR and Western blot evaluation was carried out to detect the results of Lacc1 overexpression on inflammatory responses of macrophages. As introduced in Fig. S2D & S2E, Lacc1 expression was considerably elevated within the overexpression (OE) group. In distinction, the degrees of IL-1β, IL-6, iNOS, MMP9, and MMP13 have been considerably decreased within the OE group in comparison with the NC group.
In abstract, our knowledge point out that Lacc1 performs a pivotal function in regulating each irritation and glycolysis in RAW264.7 macrophages. Thus, RAW264.7 macrophages engineered to overexpress Lacc1 might characterize a promising supply for the event of extracellular vesicles geared toward regulating metabolism and suppressing irritation.
Lacc1-Engineered EVs (OE-EVs) alleviated IL-1β induced irritation in chondrocytes
Development and characterization of OE-EVs
On this research, RAW264.7 macrophages have been transfected with a plasmid for Lacc1 overexpression. Following transfection, Lacc1-engineered vesicles (OE-EVs) have been remoted from the handled RAW264.7 macrophages utilizing ultracentrifugation, whereas the vesicles from NC plasmid-treated cells have been designated as NC-EVs.
The morphology of OE-EVs and NC-EVs was evaluated utilizing transmission electron microscopy (TEM). As illustrated in Fig. 3A, each OE-EVs and NC-EVs exhibited typical exosomal morphology, characterised by a saucer-like or hemispherical form with concave surfaces. Nanoparticle monitoring evaluation (NTA) (Fig. 3A) revealed common sizes of 126.1 nm for OE-EVs and 128.3 nm for NC-EVs, aligning with the anticipated EV measurement vary (30–200 nm). The concentrations of OE-EVs and NC-EVs have been quantified as 1.35 × 1010 particles/mL and 1.6 × 1010 particles/mL, respectively. The protein content material of OE-EVs was decided to be 0.82 mg/mL, whereas NC-EVs had 0.66 mg/mL. Western blot evaluation in Fig. 3C confirmed the presence of exosomal markers CD63, CD81, and TSG101 in each OE-EVs and NC-EVs, whereas the detrimental marker Calnexin was not detected. Moreover, Lacc1 expression was considerably greater in OE-EVs group than that within the NC-EVs group.
The uptake effectivity of OE-EVs by chondrocytes was assessed by labeling the vesicles with PKH26 dye [34]. As illustrated in Fig. 3B, each OE-EVs and NC-EVs have been successfully internalized by chondrocytes, primarily localized within the cytoplasm. PKH26 was chosen for EV labeling as a consequence of its excessive membrane affinity, robust fluorescence stability, and intensive use in EV analysis. PKH26 has been extensively utilized to trace EV uptake and biodistribution in numerous illness fashions, together with osteoarthritis and rheumatoid arthritis [35]. In comparison with different dyes, PKH26 gives low photobleaching, guaranteeing long-term visibility of labeled EVs in each in vitro and in vivo settings, making it a dependable software for monitoring EV-mediated mobile interactions. To evaluate biocompatibility, chondrocytes have been handled with numerous concentrations of OE-EVs and NC-EVs for 1, 3, and 5 days, adopted by cell viability evaluation utilizing the CCK-8 assay. As proven in Fig. S3A, chondrocytes handled with 15 µg/mL OE-EVs demonstrated the very best viability throughout all time factors. Consequently, 15 µg/mL OE-EVs and NC-EVs have been utilized in subsequent experiments. To additional assess the protecting results of EVs on chondrocytes in an inflammatory atmosphere, calcein-AM/PI dwell/useless cell staining was carried out. Fig. S3B revealed extra useless cells within the IL-1β-treated group whereas fewer useless cells have been noticed within the Con, NC-EVs, and OE-EVs teams, suggesting that each OE-EVs and NC-EVs had favorable biocompatibility and protecting results. These outcomes affirm that OE-EVs and NC-EVs have favorable biocompatibility and are effectively endocytosed by chondrocytes.
OE-EVs attenuate IL-1β-induced irritation in chondrocytes. (A) TEM and NTA characterization of NC-EVs and OE-EVs. (B) PKH26-labeled EVs internalized by chondrocytes. (C) Western blot evaluation 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 ranges measured by ELISA. (H) Western blot of inflammation- and ECM-related markers. (n = 3, *p < 0.05, **p < 0.01, and ***p < 0.001)
OE-EVs alleviated IL-1β induced irritation in chondrocytes in vitro
Diminishing irritation and selling cartilage matrix synthesis are essential for the efficient therapy of TMJ osteoarthritis. To judge the therapeutic results of OE-EVs on IL-1β induced inflammatory chondrocytes, we assessed the expression of key markers concerned in cartilage matrix formation and the inflammatory cytokines. As proven in Fig. 3D, qRT-PCR outcomes indicated a big discount within the expression of collagen II, aggrecan, and Sox9, key markers for cartilage matrix synthesis in IL-1β group. Nevertheless, therapy with OE-EVs resulted in a big upregulation of those markers, indicating that OE-EVs promote cartilage matrix synthesis underneath inflammatory situations. Furthermore, the expression of inflammatory cytokines (together with IL-1β and IL-6), and matrix metalloproteinase (together with MMP9 and MMP13) was markedly elevated following IL-1β induction, whereas OE-EVs therapy successfully downregulated these cytokines.
Western blot evaluation corroborated these findings. As illustrated in Fig. 3H, the degrees of IL-1β, IL-6, MMP3, and MMP9 have been considerably enhanced with IL-1β stimulation. Nevertheless, the OE-EVs therapy successfully counteracted the pro-inflammatory results of IL-1β. The expression of Sox9 confirmed an reverse development, additional supporting the potential of OE-EVs in selling cartilage matrix synthesis and chondrocyte anabolic metabolism.
To additional assess the affect of OE-EVs on inflammatory cytokine secretion, ELISA was carried out. Determine 3F demonstrated that OE-EVs lowered the extreme secretion of IL-1β, additional confirming their anti-inflammatory properties. As well as, immunofluorescence staining for aggrecan (Fig. 3E) and MMP13 (Fig. 3G) in chondrocytes confirmed that IL-1β stimulation decreased aggrecan expression whereas rising MMP13 expression. Conversely, OE-EVs therapy counteracted these results, leading to greater aggrecan ranges and decrease MMP13 expression.
To additional examine the mechanism by which OE-EVs modulate the inflammatory response, we examined their impact on macrophages. RT-PCR and Western blot analyses of LPS-stimulated macrophages handled with OE-EVs revealed a big downregulation of pro-inflammatory markers, together with TNF-α, MMP9, MMP13, IL-1β, and iNOS, whereas the expression of Arg1 was markedly elevated (Fig. S4). These findings point out that OE-EVs primarily operate by suppressing M1 macrophage polarization, thereby mitigating the inflammatory phenotype.
In articular cartilage, the extracellular matrix (ECM) is primarily composed of aggrecan and sort II collagen. Aggrecan performs a pivotal function in sustaining cartilage phenotype and selling chondrocyte proliferation, whereas sort II collagen, encoded by the gene COL2A1 and synthesized by chondrocytes, is a significant structural collagen [36,37,38]. Sox9, a transcription issue essential for chondrogenesis, is expressed from the multifunctional mesenchymal precursor stage to the cell differentiation stage, and its inactivation hindered the event of cartilage [39]. Due to this fact, these cartilage-specific genes are important for sustaining chondrocyte operate and programming anabolic metabolism.
Nevertheless, within the inflammatory atmosphere of OA, pro-inflammatory cytokines (comparable to IL-1β and IL-6) and matrix-degrading enzymes (comparable to MMP3, MMP9, MMP13, and ADAMTS5) promote irritation and catabolic metabolism, resulting in ECM degradation and impaired chondrocyte operate. IL-1β is considerably elevated in synovial fluid of TMJOA sufferers and mediates matrix degradation by way of inducing expression of matrix degradative enzymes comparable to MMP3, MMP9, MMP13 and ADAMTS5 [39, 40]. Apart from, elevated by irritation, extreme manufacturing of reactive metabolites, together with reactive oxygen (ROS) and reactive nitrogen species (RNS), contribute to mitochondrial dysfunction, matrix degradation, and cell injury in OA [9]. These components create a vicious cycle that exacerbates cartilage degeneration.
Earlier research have demonstrated that MSC-derived exosomes can ameliorate irritation in TMJOA by decreasing IL-1β and iNOS expression [40]. Furthermore, engineered EVs have been reported to ship purposeful molecules, comparable to miRNAs, to restore the immune microenvironment in OA joints, thereby assuaging OA development [41]. Current analysis highlights the essential function of synovial macrophages in OA pathogenesis, as they secrete pro-inflammatory cytokines that work together with chondrocytes and exacerbate cartilage degradation [14]. Moreover, macrophage-derived EVs have been implicated in cartilage catabolism and synovial irritation, additional underscoring their contribution to OA development [42]. In keeping with these findings, our outcomes show that OE-EVs not solely shield cartilage ECM homeostasis by enhancing matrix synthesis and inhibiting degradation but additionally mitigate chondrocyte irritation in vitro. These findings strongly assist the potential of OE-EVs as a novel metabolic and immunomodulatory remedy for OA, paving the way in which for future investigations into their function in regulating power metabolism.
OE-EVs reprogrammed chondrocytes’ power metabolism by assuaging mitochondria dysfunction
Power metabolism is an important mediator of mobile operate and is usually altered throughout illness states, significantly in response to inflammatory stimuli in chondrocytes. Earlier research have demonstrated the therapeutic results of OE-EVs on chondrocytes in vitro. To additional examine the metabolic modifications in IL-1β-induced inflammatory chondrocytes, gene and protein expression associated to key enzymes concerned glycolysis have been analyzed by qRT-PCR and Western blotting. As proven in Fig. 4A and B, IL-1β therapy resulted in a big improve within the expression of HK1, HK2, LDHA, PKM2 and hypoxia-inducible issue 1-alpha (Hif-1α), indicating enhanced glycolysis exercise in OA chondrocytes. Notably, therapy with OE-EVs successfully counteracted these modifications.
Mitochondria play a central function in ATP synthesis and mobile metabolism. Irritation-induced mitochondrial dysfunction is characterised by impaired ATP manufacturing. To evaluate the affect of OE-EVs on mitochondria dysfunction induced by irritation, we carried out cell ATP assay. As illustrated in Fig. 4C, IL-1β considerably lowered ATP degree in chondrocytes, whereas OE-EVs therapy successfully restored the ATP degree.
Furthermore, we measured the extracellular lactate ranges within the supernatants of varied teams. As depicted in Fig. 4D, lactate focus was markedly elevated within the IL-1β group, whereas OE-EVs therapy led to a big discount. Moreover, mitochondrial dysfunction is characterised by an aberrant change within the mitochondrial membrane potential (ΔΨm). JC-1 staining (Fig. 4E) revealed that IL-1β-treated chondrocytes exhibited elevated inexperienced fluorescence from JC-1 monomers (indicating ΔΨm loss), whereas OE-EVs therapy resulted in outstanding crimson fluorescence from JC-1 aggregates, suggesting ΔΨm restoration.
Extreme ROS manufacturing contributes to oxidative stress and mitochondrial dysfunction, exacerbating mobile injury. DCFH-DA staining (Fig. S5) demonstrated a big improve in intracellular ROS ranges within the IL-1β group, which have been successfully lowered by OE-EVs therapy. We additionally evaluated mitochondrial ROS ranges utilizing MitoSOX staining and examined mitochondrial morphology with MitoTracker Inexperienced staining. As proven in Fig. 4F, inflammatory chondrocytes uncovered to IL-1β exhibited impaired mitochondrial morphology, together with mitochondrial deformation, swelling. Conversely, OE-EVs therapy mitigated these detrimental results. Mitochondrial ROS (mtROS) degree considerably elevated in IL-1β group, implying an escalation in mitochondrial oxidative stress injury, nevertheless, remarkably inhibited with OE-EVs therapy. As proven in Fig. 4G, the IL-1β group displayed severely broken mitochondrial buildings, comparable to lowered crista, disrupted membranes, and a swollen phenotype, in comparison with Management group. In distinction, chondrocytes handled with OE-EVs maintained comparatively regular mitochondrial morphology together with displaying elongated, rod-shaped buildings with improved cristae and membrane integrity in comparison with each IL-1β and NC-EVs teams.
In abstract, our outcomes point out that OE-EVs reprogrammed glycolytic metabolism in OA chondrocytes by inhibiting cardio glycolysis and assuaging oxidative stress, whereas concurrently enhancing mitochondrial morphology and performance. Our findings point out that Lacc1-enriched EVs regulate each chondrocyte metabolism and irritation; nevertheless, whether or not Lacc1 itself performs a direct function in chondrocyte metabolic homeostasis stays unclear. On condition that EV cargo exerts organic results by way of intercellular communication, it’s potential that the noticed metabolic modifications are mediated by way of a number of signaling pathways reasonably than direct intracellular Lacc1 exercise. Future research involving direct overexpression or knockdown of Lacc1 in chondrocytes will probably be wanted to find out its potential cell-autonomous operate. Clarifying this mechanism will probably be important for totally elucidating the therapeutic potential of Lacc1-based EV interventions in TMJOA.
OE-EVs reprogram metabolism in chondrocytes. (A&B) qRT-PCR and Western blot evaluation of glycolytic enzyme expression. (C&D) ATP manufacturing and lactate ranges in chondrocytes. (E) JC-1 staining of mitochondrial membrane potential (crimson: JC-1 aggregates, inexperienced: JC-1 monomers). (F) Mitochondrial ROS evaluation utilizing MitoSOX and Mitotracker staining. (G) TEM photographs of mitochondria in numerous teams. (n = 3, *p < 0.05, **p < 0.01, and ***p < 0.001)
RNA-Seq evaluation of the affect of OE-EVs on glycolysis and irritation in OA chondrocytes
To additional discover the underlying mechanism behind the therapeutic results of OE-EVs, transcriptomic evaluation was carried out. A heatmap (Fig. 5A) depicting differentially expressed genes (DEGs) in IL-1β and OE-EVs-treated teams revealed important alterations within the expression of key regulators of glycolytic and inflammatory. Notably, within the IL-1β group, the expression of glycolytic genes, together with HK2, Pfkfb3, and Eno3, was considerably upregulated, suggesting enhanced glycolytic metabolism (Fig. 5A). In distinction, OE-EVs therapy markedly downregulated the expression of those genes (Fig. 5A), indicating a possible function of OE-EVs in glycolytic reprogramming in chondrocytes.
Moreover, inflammatory genes, comparable to Cxcl5, Ccl2, Nfkbia, Tnfaip3, and IL6, have been considerably downregulated within the OE-EVs group in comparison with the IL-1β group. This downregulation additional helps the anti-inflammatory results of OE-EVs in OA chondrocytes. Furthermore, the expression of genes concerned in ECM degradation, together with MMP3 and Adamts18, was considerably lowered within the OE-EVs-treated group, suggesting that OE-EVs could play a task in restraining ECM breakdown in OA. As well as, the expression of JAK2, a key element of the JAK-STAT signaling pathway, was notably decreased following OE-EVs therapy, additional suggesting that OE-EVs could modulate glycolysis and irritation, whereas suppressing ECM degradation and selling proteoglycan synthesis in OA chondrocytes.
To realize additional insights into the organic processes underpinning these modifications, Gene Ontology (GO) enrichment evaluation was carried out. As proven in Fig. 5C, probably the most considerably enriched GO phrases included processes associated to glycolytic metabolism, carbohydrate metabolism, ATP era from ADP, extracellular matrix group, and glycosaminoglycan binding. These findings spotlight the potential of OE-EVs in regulating metabolic processes and sustaining ECM homeostasis in OA.
In assist of those outcomes, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway evaluation (Fig. 5B) recognized a number of enriched pathways, together with HIF-1 signaling, glycolysis/gluconeogenesis, TNF signaling, and JAK-STAT signaling. These pathways are per OE-EVs’ function in modulating glycolytic metabolism and irritation. As well as, Gene Set Enrichment Evaluation (GSEA) revealed notable variations in JAK-STAT signaling pathway and extracellular matrix (Fig. 5D), TNF signaling pathway and cell adhesion (Fig. S6). In comparison with the IL-1β group, the enrichment of genes related to extracellular matrix and cell adhesion was considerably elevated within the OE-EVs group, in the meantime the genes regarding JAK-STAT and TNF signaling pathway displayed a big lower within the OE-EVs group. These outcomes counsel that OE-EVs not solely promote ECM synthesis but additionally inhibit the inflammatory response, seemingly by way of modulation of the JAK-STAT signaling pathway.
Earlier research have proven that pro-inflammatory cytokines (comparable to IL-1β, IL-6) and matrix metalloproteinase (MMP) activate the Janus Kinase/Sign Transducers and Activators of Transcription (JAK/STAT) signaling pathway, contributing to irritation and cartilage degradation in OA. Inhibition of the JAK-STAT pathway has been proven to alleviate chondrocyte irritation and shield towards cartilage degeneration [43, 44]. To additional confirm the correlation between anti-inflammation results of OE-EVs with regulating JAK-STAT signaling, we carried out western blot evaluation. As proven in Fig. 5E and F, the degrees of phosphorylated JAK2 (p-JAK2) and STAT3 (p-STAT3), markers of JAK-STAT activation, in addition to the ratios of p-JAK2/JAK2 and p-STAT3/STAT3, have been considerably lowered within the OE-EVs group in comparison with the IL-1β group. These outcomes point out that OE-EVs exert inhibitory results on the JAK-STAT pathway, thereby suppressing irritation in OA chondrocytes.
In abstract, our findings counsel that OE-EVs play an lively function in regulating irritation microenvironment of TMJOA. By reprogramming glycolysis, inhibiting key inflammatory pathways (together with JAK-STAT, TNF, and HIF-1 signaling), and selling ECM synthesis, OE-EVs assist create a positive microenvironment for chondrocyte operate and shield towards cartilage degradation.
Transcriptomic and signaling pathway evaluation of IL-1β- and OE-EV-treated chondrocytes. (A) Heatmap of differentially expressed genes. (B&C) KEGG and GO enrichment evaluation. (D) GSEA evaluation of key pathways. (E&F) Western blot and semi-quantification of JAK-STAT signaling. (n = 3, *p < 0.05 and **p < 0.01)
OE-EVs alleviate irritation and promote cartilage restore in TMJOA in vivo
Constructing upon the promising in vitro outcomes, the therapeutic results of OE-EVs have been additional assessed in vivo utilizing a surgical induced mouse mannequin of TMJOA [45, 46]. As illustrated in Fig. 6A, therapies started two weeks after unilateral discectomy to permit for TMJOA growth. Mice in TMJOA group acquired intra-articular injections of PBS, whereas the NC-EVs and OE-EVs teams acquired NC-EVs and OE-EVs, respectively. 4 weeks post-treatment, TMJ tissues have been harvested for evaluation.
To look at the retention capacity of EVs in vivo, PKH26 labeled OE-EVs and NC-EVs have been injected into the TMJ, and fluorescence alerts have been tracked utilizing an in vivo imaging system (Berthold, Germany). Fig. 6B confirmed that each OE-EVs and NC-EVs have been retained within the tissue for over 72 h. For assurance of the sustained results of OE-EVs, intra-articular injection was administered twice weekly. TMJs have been harvested after 4 weeks of therapy.
Since irritation in TMJOA additionally results in subchondral bone deterioration, we consider the therapeutic results of OE-EVs on subchondral bone transforming utilizing Micro-CT and parametric evaluation. The Micro-CT reconstruction photographs of condyle in Fig. 6C confirmed extreme condylar cartilage and subchondral bone destruction together with irregular trabecular buildings and typical subchondral bone loss within the TMJOA group. In distinction, OE-EVs therapy notably preserved subchondral bone integrity, presenting a extra common trabecular sample in comparison with the TMJOA and NC-EVs teams.
Subsequently, Bone structural parameters—together with the ratio of bone quantity to tissue quantity (BV/TV), trabecular thickness (Tb.Th), trabecular quantity (Tb.N) and trabecular separation (Tb.Sp) have been additionally examined. As proven in Fig. 6D, the TMJOA group had considerably decrease BV/TV, Tb.Th, and Tb.N, together with elevated Tb.Sp, indicating substantial bone construction deformation. OE-EVs therapy considerably improved BV/TV, Tb.Th, and Tb.N whereas decreasing Tb.Sp, suggesting an efficient amelioration of bone loss. No important variations have been noticed between the TMJOA and NC-EVs teams. Thus, these outcomes advised that OE-EVs therapy successfully alleviated subchondral bone erosion attributable to TMJOA and guarded condylar subchondral bone construction.
OE-EVs exhibit glorious biocompatibility and don’t trigger systemic toxicity. To additional assess the security of OE-EVs in vivo, we carried out H&E staining of main organs, together with the guts, liver, spleen, lungs, and kidneys. The histological evaluation (Fig. S7) revealed no detectable pathological abnormalities throughout all teams, indicating that OE-EV therapy doesn’t induce observable toxicity or tissue injury. These findings, along with our in vitro CCK-8 and dwell/useless staining assays (Fig. S3), affirm that OE-EVs are well-tolerated and biocompatible, supporting their potential for medical software.
Histological evaluation was additional carried out to guage modifications in articular cartilage and subchondral bone. H&E staining (Fig. 6E) revealed attribute TMJOA pathology within the TMJOA group, together with disorganized mobile layers, a tough floor, inflammatory cells infiltration, and extreme subchondral bone injury, whereas the OE-EVs group exhibited lowered TMJOA associated histological alterations in comparison with the TMJOA and NC-EVs teams. Furthermore, the Safranin O-fast inexperienced staining (Fig. 6F) was carried out to evaluate the glycosaminoglycan (GAG) degree deposited in cartilage, with TMJOA cartilage exhibiting diminished and irregular Safranin O+ matrix in comparison with the Con group. Notably, OE-EVs therapy resulted in a uniform and ample distribution of the safranin O+, suggesting enhanced GAG synthesis. Moreover, TB staining (Fig. 6G) revealed marked cartilage loss within the TMJOA group, whereas OE-EVs therapy preserved cartilage thickness, indicating efficient safety towards cartilage matrix degradation. Semi-quantitative evaluation of toluidine blue-stained cartilage space in Fig. S8 confirmed a 1.55-fold improve within the OE-EV group in comparison with the TMJOA group, indicating enhanced proteoglycan accumulation. Earlier research reported that EVs or engineered EVs restore TMJ condylar construction with amelioration in cell association, cartilage thickness, cellularity and matrix synthesis and subchondral bone transforming [47, 48]. In step with healing results as earlier work reported, OE-EVs therapy exhibited outstanding therapeutic results in restoration of the TMJ condylar construction, together with enhancements in morphology and purposeful elements.
OE-EVs promote cartilage restore and suppress irritation in TMJOA in vivo. (A) Schematic of OE-EV therapy in a TMJ-OA mouse mannequin. (B) In vivo fluorescence imaging of TMJ-injected OE-EVs and NC-EVs. (C&D) Micro-CT photographs and bone structural parameters. (E–G) Histological staining (H&E, Safranin O-Quick Inexperienced, Toluidine Blue). (n = 6, *p < 0.05, and ***p < 0.001)
Collagen II and Aggrecan are important for chondrogenesis, and their expression ranges have been evaluated by immunohistochemistry and immunofluorescence staining, respectively. Collagen II (Fig. 7A) and Aggrecan (Fig. 7C and F) expression was considerably suppressed in TMJOA group in comparison with the Con group. Whereas OE-EVs markedly enhanced their expression, indicating that OE-EVs therapies successfully promote cartilage matrix regeneration and anabolic exercise.
Furthermore, inflammatory cytokines (comparable to IL-1β) and matrix metalloproteinases (MMP3, MMP9, MMP13) performed pivotal roles in cartilage matrix degradation. Herein, the affect of OE-EVs on assuaging cartilage irritation was investigated by immunohistochemical staining for IL-β (Fig. 7B) and immunofluorescence staining for MMP13 (Fig. 7D and G) and MMP3 (Fig. 7E and H). Because the outcomes confirmed, the expression of those proteins was considerably elevated within the TMJOA group; nevertheless, OE-EVs therapy successfully lowered IL-1β, MMP3 and MMP13 ranges, demonstrating that OE-EVs considerably suppress catabolic and pro-inflammatory components. As Fig. 7I confirmed, histological evaluation was carried out utilizing the OA Analysis Society Worldwide (OARSI) scoring system. The OE-EVs group displayed a considerably decrease rating in comparison with the TMJOA group, indicating a considerable therapeutic impact in mitigating OA-related injury.
We additionally carried out immunofluorescence staining of iNOS and Arg1 to confirm the anti-inflammatory results of OE-EVs in vivo. As Fig. S9 confirmed, these outcomes demonstrated that within the OE-EV-treated group, Arg1 expression was upregulated, whereas iNOS expression was considerably lowered. This implies that OE-EVs could mitigate TMJOA irritation by selling macrophage polarization towards an anti-inflammatory M2 phenotype, additional supporting their therapeutic potential in modulating the joint microenvironment.
Primarily based on the above outcomes, OE-EVs exhibited favorable therapeutic potential by attenuating irritation, enhancing cartilage regeneration, rebalancing cartilage anabolism and catabolism, stopping cartilage matrix degradation, and transforming subchondral bone. Total, OE-EVs efficiently promoted cartilage restore and alleviated TMJOA irritation development in vivo.
Whereas our 4-week knowledge show the capability of OE-EVs to mitigate early-stage TMJOA irritation, persistent illness modification requires analysis over prolonged intervals (≥ 8 weeks). Future research will optimize dosing schedules and incorporate longitudinal imaging (e.g., in vivo micro-CT) to trace OA development dynamically. That is vital for medical translation, as TMJOA therapies should show sturdiness past acute symptom aid.
Whereas our TMJOA animal mannequin gives priceless insights into illness mechanisms, it might not totally replicate the complexity of human TMJOA. Notably, anatomical and physiological variations exist between species, and animal fashions usually simulate solely particular elements of the human situation [4]. Moreover, the long-term security and stability of engineered extracellular vesicles (OE-EVs) stay areas requiring additional investigation. Elements comparable to optimum storage situations and potential immunogenic responses have to be totally evaluated to make sure their viability as therapeutic brokers.
Histological and immunohistochemical evaluation 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 depth. (I) OARSI scoring for cartilage degradation. (n = 3, **p < 0.01, and ***p < 0.001)
