The availability of human cell-based models capturing molecular processes of cartilage degeneration can facilitate development of disease-modifying therapies for osteoarthritis [1], a currently unmet clinical need. Here, by imposing specific inflammatory challenges upon mesenchymal stromal cells at a defined stage of chondrogenesis, we engineered a human organotypic model which recapitulates main OA pathological traits such as chondrocyte hypertrophy, cartilage matrix mineralization, enhanced catabolism and mechanical stiffening. To exemplify the utility of the model, we exposed the engineered OA cartilage organoids to factors known to attenuate pathological features, including IL-1Ra, and carried out mass spectrometry-based proteomics. We identified that IL-1Ra strongly reduced production of the transcription factor CCAAT/enhancer-binding protein beta [2] and demonstrated that inhibition of the C/EBPβ-activating kinases could revert the degradative processes. Human OA cartilage organoids thus represent a relevant tool towards the discovery of new molecular drivers of cartilage degeneration and the assessment of therapeutics targeting associated pathways.

Articular cartilage (AC) damage heals poorly and can progress to osteoarthritis. Implantation of AC fragments (Minced Cartilage Implantation, MCI) is a promising one-step repair technique but is constrained by the limited availability of healthy AC. In this study, we evaluated the feasibility of MCI using nasal septal cartilage (NSC) as an alternative source of hyaline tissue with strong regenerative capacity. NSC obtained from rhinoplasties was decontaminated using a novel protocol, minced with or without Poloxamer 188 (P188), embedded in collagen I gel (0.5 mL per sample), and cultured for 42 days in platelet-rich plasma (PRP)-supplemented medium. The decontamination procedure with a combination of antibiotics was effective and did not impair cell viability. Histology of the resulting constructs confirmed robust cellular outgrowth and matrix deposition. Tissues produced from NSC and fragmented with P188 contained more cartilaginous matrix than those from NSC fragmented without P188 and those from AC fragmented with P188. NSC fragments embedded in a 1 mL hydrogel, sufficient for clinically relevant defect volumes, also demonstrated strong outgrowth and satisfactory matrix formation. Overall, the developed protocol supports the use of NSC as a viable tissue source in gel-based, injectable MCI grafts for focal cartilage repair.

Osteoarthritis (OA) is a multi-etiological disorder characterised by cartilage degradation, synovial inflammation, and compromised joint integrity. Current OA treatments fail to promote cartilage regeneration, often culminating in joint replacement during advanced stages. Nasal chondrocytes (NCs) have shown promise in cartilage repair and are undergoing clinical trials for focal knee cartilage defects. However, the chronic inflammatory environment in OA joints, driven by synovial-derived proinflammatory cytokines, hinders cartilage regeneration. Moreover, the mechanisms underlying OA pathogenesis, including synovium-cartilage interplay, remain poorly understood due to limitations of traditional in vitro models. This study addresses these gaps through two approaches: (1) evaluating anti-inflammatory cytokines to mitigate OA-associated synovial inflammation and engineer NC-based chondrogenic tissues expressing most effective anti-inflammatory cytokine; (2) assessing anti-inflammatory properties of engineered tissues through coculture with patient-derived OA synovial organoids and OA synovial fluid (OASF) and their regenerative potential by coculturing the engineered tissues with osteoarthritic cartilage organoids.Synovial explants from knee arthroplasty patients were cultured under chronic inflammatory conditions mimicking OA, using IL-6, IL-1β, and TNFα. Synovial fluid samples were collected from OA patients (N=54). Anti-inflammatory cytokines (IL-1Ra, IL-37, IL-1R2, IL-10) were tested for their ability to reduce synovial inflammation markers using RT-qPCR. NCs were genetically modified via lentiviral transduction to express IL-1Ra and cultured as 3D microaggregates in a defined chondrogenic medium. Patient-derived OA synovial organoids (P0-Syn) while others were established using cocultures of fibroblast-like synoviocytes (FLS) and immune cells to mimic OA pathology. Micro-cartilage spheroids derived from bone marrow stem cells (BMSCs) were exposed to inflammatory conditions, including P0-Syn conditioned media (P0-Syn CM), assessed for inflammatory and catabolic markers.IL-1Ra demonstrated the highest anti-inflammatory potency, significantly reducing synovial pro-inflammatory markers. IL-1Ra transduced NC secreted IL-1Ra sustainably and produced cartilaginous tissue with type II collagen amounts comparable to those of untransduced NC. Cartilage organoids exposed to P0-Syn CM exhibited increased matrix degradation, with elevated MMP13 expression and inflammatory markers, resembling OA pathology. OASF samples were stratified and pooled as high IL6 (median: 1670.14 pg/ml) and low IL6 (median: 83.12 pg/ml) inflammatory samples for subsequent experiments.IL-1Ra-NCs can produce high-quality cartilaginous tissues and sustainably release IL-1Ra. Patient-derived OA synovial organoids and BMSC-derived cartilage organoids are viable models for testing anti-OA therapeutics. Studies are now ongoing to test the anti-inflammatory and pro-anabolic effects of IL-1Ra transduced NCs using these models, as well as their ability to withstand OA synovial fluid-mediated inflammatory insult.