Osteoarthritis (OA) is the most common chronic disease. It is characterized by high levels of clinical heterogeneity and low inflammation. Therefore, elucidation of the mechanisms that regulate gene expression is critical for developing effective OA therapies. This study aimed to explore the role of LKB1/AMPK in the progression of OA. Anterior cruciate ligament transection (ACLT) was performed on Sprague Dawley (SD) rats right knee to construct OA model, followed by AICAR [AMP-activated protein kinase (AMPK) activator] treatment. The level changes [AMPK, IL-10, IL-13, IL-1β, TNF-α, IL-6, ASC, Caspase-1, Ki67, and hibit Nod-like receptor protein 3 (NLRP3)] and the degree of tissue injury were assessed by western blot, Immunohistochemical (IHC), Enzyme-linked immunosorbent assay (ELISA), Hematoxylin-eosin staining (HE), Immunofluorescence (IF), Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, and Safranin O and Fast Green staining (S–O). Human chondrocytes were induced by LPS to construct a cellular inflammatory model, and then transfected with oe-AMPK or oe-HOIL-1–interacting protein (HOIP). Cell viability/apoptotic and intracellular content of AMPK, HOIP, IL-1β, IL-10, IL-13, TNF-α, IL-6, ASC, NLRP3 and Caspase-1 were measured by western blot, ELISA, CCK-8, IF, flow cytometry and TUNEL assays. After AICAR treatment with OA rats, the expression of p-AMPK, IL-10, IL-13, Ki67 and Bcl-2 increased, the level of NLRP3 inflammasome, TNF-α, IL-6, Bax and Caspase-3 levels were decreased, and tissue damage and apoptosis were significantly alleviated. After transfected with oe-LKB1, chondrocyte activity and LKB1 linear ubiquitination were enhanced, and the level of HOIP, p-AMPK, IL-10 and IL-13 were increased. In contrast, NLRP3 inflammasome (ASC, NLRP3, Caspase-1, IL-1β, and cleaved Caspase-1), TNF-α, and IL-6 levels decreased, apoptosis rate and TUNEL positive rate were attenuated. LKB1/AMPK pathway significantly ameliorated NLRP3 inflammasome response and chondrocyte injury. Activation of AMPK pathway by linear ubiquitination of LKB1 may be a potential target for OA treatment. This study highlights the importance of the LKB1/AMPK pathway in NLRP3 inflammatory body response and chondrocyte injury. Activation of LKB1 by modulating linear ubiquitination may be a potential target for OA treatment.

Senescent cells exert a significant influence over their surrounding cellular environment. Senescent chondrocytes (SnChos) were found to be accumulated in degenerated cartilage present in joints affected by osteoarthritis. The influence of SnChos on exogenously transplanted stem cells has yet to be reported. In this study, we evaluated the interactions between SnChos and bone marrow mesenchymal stem cells (BMSCs) when co-cultured as well as in the intra-articular senescent microenvironment (IASM). The effect of IASM on cartilage regeneration was also assessed. It was found that a small fraction of SnChos induced BMSC cellular senescence and apoptosis. SnChos also inhibited proliferation, facilitated stemness, and suppressed chondrogenic differentiation of BMSCs. BMSCs induced the apoptosis of SnChos, reduced the proportion of SnChos, stimulated SnChos proliferation, and revealed a bidirectional effect on SnChos inflammaging. IASM significantly suppressed the survival, proliferation, and appropriate differentiation of grafted BMSCs in vivo, all of which impaired cartilage regeneration. Anti-senescence agent ABT-263 was able to partly rescue the cells from the negative effects of SnChos. The SnChos and BMSCs interacted with each other at cellular senescence, apoptosis, proliferation, differentiation, and cell functions. This interaction impaired the cartilage repair of MSCs. Anti-senescence agent provided a possible solution for this impairment.

Direct intra-articular injection (DIAI) of mesenchymal stem cells (MSCs) is a promising technique for cartilage repair. However, the repair process was hindered by the absence of scaffold and poor cell–matrix interactions. In this study, we developed a pericellular collagen I coating (PCC) on MSCs. The overall performances of MSC-PCC homing, chondrogenic differentiation, and cartilage regeneration have been comprehensively evaluated in a New Zealand rabbit model. Firstly, we examined the morphology and physical characteristics of PCC. Secondly, MSC ex-vivo cartilage slice adhesion and in-vivo cartilage defect homing were observed using multiscale methods. Thirdly, the precartilage condensation of cell pellets formed by aggregation of MSCs was examined to evaluate the cartilage-inducing potential of PCC. Finally, the cartilage regeneration by DIAI of PCC-coated MSCs was observed and scored macroscopically and histologically. In general, the cell adhesion and homing assay revealed that PCC facilitated MSC adhesion on cartilage slices, enhancing MSC homing and retention to cartilage defect. This increased homing ratio was accompanied by an increasing cell–cell contact. Compared with naked MSCs, the cell pellets formed by PCC-coated MSCs exhibited more evident appearance of condensation. In pellets, cell–cell interaction has been significantly stimulated, inducing the expression of condensation marker N-cadherin, and subsequent chondrogenic marker collagen II and aggrecan. By 12 weeks after DIAI, cartilage defects have been repaired by MSCs to varying degrees. Overall, PCC significantly enhances the quality of cartilage regeneration judging from macroscopic observation, ICRS score, histological examination, and collagen type I, II, and X immunohistochemical staining. The capacity and viability of MSCs can be enhanced by collagen I coating, which provides cues for enhancing cell homing and differentiation. Our method provides a novel strategy for stem cell therapy.

The heterogeneity of osteoarthritis (OA) significantly limits the effectiveness of pharmacological treatments in an unselected patient population. In this context, the identification of OA subtypes is meaningful for the development of therapies that target specific types of OA pathogenesis. Expression array profiles of 70 OA and 36 control synovial samples were extracted from the GEO database. Unsupervised consensus clustering was performed based on the most variable genes to identify OA subclusters. Next, Joint samples from OA patients were obtained. We divided the OA patient into two subpopulations according to synovial ADCY7 levels. Synovium and cartilage samples from different OA subpopulations were evaluated. In addition, we established a high-fat diet (HFD)-induced rat OA model. We evaluated OA progression, lipid metabolism, synovitis and fibroblast-like synoviocytes (FLS) function in this HFD-induced OA model. 70 OA patients were categorized into three distinct subclusters. We noted that one subcluster was characterized by synovial lipid metabolism disorder GO terms. We further identified the most noticeable KEGG pathway “Regulation of lipolysis in adipocytes” in this subcluster as well as the most significantly differentially expressed gene, ADCY7. We found that the ADCY7 high expressing group (32.6%) exhibited features of synovial inflammatory lipolysis epithelial-mesenchymal transition (EMT) tendency, as well as faster join space narrowing. The HFD induced OA-like degeneration in rat joints. We observed similar synovial inflammatory lipolysis and EMT in FLS, characterized by higher proliferative and invasive activity and elevated proinflammatory and procatabolic properties. ADCY7 was highly expressed in the synovium of the HFD-OA model rats and the inhibition of ADCY7 effectively attenuated these HFD-induced degenerative changes as well as synovial inflammatory lipolysis and FLS dysfunction. In HFD-FLSs, ADCY7 promoted the phosphorylation of PKA as well as its downstream lipid droplet-associated protein PLIN1 and hormone-sensitive lipase (HSL). The inhibition of PKA largely alleviated ADCY7-mediated HFD-FLS dysfunction. We described a synovial EMT and lipid metabolism disorder in the pathogenesis of OA. This novel mechanism may represent a currently undefined OA subtype. ADCY7 is a potential molecular marker of this pathomechanism. Utilizing synovial samples from OA patients, we identified a subpopulation with high ADCY7 expression. This may represent a currently undefined OA subtype and explain the clinical phenomenon of more severe synovial inflammation in obese OA patients. In addition, we established an HFD-induced OA rat model and found an upregulation of ADCY7 in the synovium. We confirmed that the inhibition of ADCY7 could effectively attenuate HFD-induced degenerative changes as well as the inflammatory lipolysis and FLS dysfunction observed in the rat model. This suggests that ADCY7 and its downstream pathways are potential pharmacological targets for treating this lipid-metabolism-disorder-related OA mechanism.

Background: Macrophage is a central regulator of innate immunity. Its M2 subsets, such as interstitial synovial macrophages, have been found to play critical roles in suppressing chronic inflammation and maintaining homeostasis within the joint. These macrophages have great potential as a disease-modifying cell therapy for osteoarthritis (OA). However, this has not yet been studied.Methods: Macrophages were isolated from the bone marrow of rats. We constructed a stable macrophage that “locked” in anti-inflammatory and pro-regenerative M2a polarity (L-M2a) by simultaneously knocking out tumor necrosis factor receptor 1 (TNFR1) and overexpressing IL-4 using Cas9-ribonuclear proteins (Cas9-RNP) and electroporation. In vitro, these L-M2a macrophages were treated with OA synovial fluid or co-cultured with OA chondrocytes or fibroblast-like synoviocytes (FLS). In vivo, L-M2a macrophages were injected intra-articularly to evaluate their homing and engrafting abilities and therapeutic effects on OA progression using a rat model.Results: L-M2a macrophages displayed a typical anti-inflammatory phenotype similar to that of M2 macrophages in vitro. In OA microenvironment, L-M2a macrophages maintained a stable anti-inflammatory phenotype, whereas unmodified M2 macrophages lost their phenotype and switched to M1 polarity. L-M2a macrophages demonstrated a potent anti-inflammatory effect in crosstalk with OA-FLSs and an anti-degenerative effect in crosstalk with senescent OA chondrocytes. In vivo, compared with M2 macrophages and exosomes, L-M2a macrophages exhibited significantly superior therapeutic effects in OA by successfully resolving inflammation, restoring tissue homeostasis, and promoting cartilage regeneration.Conclusion: The engineered L-M2a macrophages maintained a superior anti-inflammatory and pro-regenerative capacity in the inflammatory OA microenvironment and represents an ideal new strategy for the disease-modifying therapy of OA.

Three-dimensional (3D) bioprinting is an advanced tissue engineering technique that has received a lot of interest in the past years. We aimed to highlight the characteristics of articles on 3D bioprinting, especially in terms of research hotspots and focus. Publications related to 3D bioprinting from 2007 to 2022 were acquired from the Web of Science Core Collection database. We have used VOSviewer, CiteSpace, and R-bibliometrix to perform various analyses on 3,327 published articles. The number of annual publications is increasing globally, a trend expected to continue. The United States and China were the most productive countries with the closest cooperation and the most research and development investment funds in this field. Harvard Medical School and Tsinghua University are the top-ranked institutions in the United States and China, respectively. Dr. Anthony Atala and Dr. Ali Khademhosseini, the most productive researchers in 3D bioprinting, may provide cooperation opportunities for interested researchers. Tissue Engineering Part A contributed the largest publication number, while Frontiers in Bioengineering and Biotechnology was the most attractive journal with the most potential. As for the keywords in 3D bioprinting, Bio-ink, Hydrogels (especially GelMA and Gelatin), Scaffold (especially decellularized extracellular matrix), extrusion-based bioprinting, tissue engineering, and in vitro models (organoids particularly) are research hotspots analyzed in the current study. Specifically, the research topics “new bio-ink investigation,” “modification of extrusion-based bioprinting for cell viability and vascularization,” “application of 3D bioprinting in organoids and in vitro model” and “research in personalized and regenerative medicine” were predicted to be hotspots for future research.