Background/Purpose: Our ultimate goal is to study potential drug candidates in an experimental setting of arthritis. Therefore, we aim to develop a human in vitro 3D joint model mimicking key features of arthritis by applying inflammatory conditions namely immune cells and pro-inflammatory cytokines. Our in vitro 3D joint model consists of different components: i) osteogenic and ii) chondrogenic part, iii) joint space with synovial fluid and iv) synovial membrane. Developed as an alternative in vitro approach to animal experiments, our 3D joint model will enable us to study efficiently the effects of potential drug candidates in a more translational setup.

Here, we aimed to demonstrate the suitability of our human in vitro 3D osteochondral tissue model (OTM) by analyzing the influence of the main cytokines involved in the pathogenesis of RA as well as the impact of approved drugs.

Methods: The OTM was engineered by co-cultivation of human mesenchymal stromal cell (hMSC)-derived bone and cartilage components in a 3D environment and comprehensively characterized (e.g. cell vitality, morphology, structural integrity) using histological, biochemical and molecular biological methods, µCT and scanning electron microscopy. In brief, to establish the osteogenic component, we populated β-tricalcium phosphate (TCP) – mimicking the mineral bony part – with hMSCs, while the scaffold-free cartilage component was generated by cellular self-assembly and intermittent mechanical stimulation (fzmb GmbH). To test the suitability of our OTM, we applied a cocktail of TNFα, IL-6 and MIF using concentrations reported from RA synovial fluid alone or in combination with approved therapeutic drugs and analyzed their impact by qPCR.

Results: We verified the osteogenic phenotype of our 3D bone component by demonstrating an increase in mineralized bone volume and the induction of bone-related gene expression (RUNX2, SPP1, COL1A1, OC) as compared to the corresponding control. Secondly, we verified the chondrogenic phenotype of our cartilage component by HE and Alcian Blue staining as well as by the reduced expression of COL1A1 and an abundant expression of COL2A1. Interestingly, co-cultivation of both components for up to 3 weeks demonstrated colonization, connectivity and initial calcification implying a transitional bridging area. The exposure of OTM to TNFα, IL-6 and MIF caused cell- and matrix-related changes, such as the significant induced expression of the metabolic marker LDHA, the angiogenic marker VEGF and the inflammation markers IL8 and TNF in both bone and cartilage, while IL6 is downregulated in bone compared to the unstimulated control. Moreover, a cytokine-related significant upregulation of MMP1 and MMP3 expression was observed in cartilage compared to bone. Due to the specific drug treatment (adalimumab, tocilizumab, milatuzumab), the induction of inflammation and degradation could be prevented.

Conclusion: The results of our study showed that our human in vitro 3D OTM mimics cytokine-driven cell- and matrix-related changes – key features of RA. By combining the components in a 96-well format, we aim to provide a mid-throughput system for preclinical drug screening.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mimicking-cytokine-driven-key-features-of-arthritis-using-a-human-in-vitro-3d-joint-model/