FRI0507 THE HUMAN-BASED IN VITRO 3D ARTHRITIC JOINT MODEL
Authors: Alexandra Damerau, Annemarie Lang, Moritz Pfeiffenberger, Timo Gaber, Frank Buttgereit
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background</h3> Our ultimate goal is to develop a valid human <i>in vitro</i> 3D joint model to simulate the pathogenesis of arthritis. The model consists of different components including an osteogenic and chondrogenic part, the joint space with synovial fluid and the synovial membrane. It contains all involved cell types and thus, to allow interactions between cells by cell contacts and signaling molecules. The arthritic joint is simulated by the application of sorted immune cells and typical cytokines. As an alternative experimental setup for animal models, our <i>in vitro</i> 3D joint model will enable us to study the influence and efficacy of potential drug candidates. Currently, there is no valid 3D model, which is able to mimic an arthritic joint. <h3>Objectives</h3> Here, we aimed to develop the single joint components, namely the osteogenic and chondrogenic part, the joint space with synovial fluid and the synovial membrane, separately and in co-culture without and with the application of neutrophils (PMNs) and cytokines. <h3>Methods</h3> We have used human bone marrow derived mesenchymal stromal cells (hMSCs) to develop the different 3D tissue components that are characterized in detail (e.g. cell vitality, structural integrity) using histological, biochemical and molecular biological methods as well as μCT and scanning electron microscope (SEM). For the osteogenic component, we populated β-tricalcium phosphate (TCP) – mimicking the mineral bony part – with hMSCs, while the scaffold-free 3D cartilage component was generated from a marrow tissue exclusively consisting of hMSC under intermittent mechanical stimulation (fzmb GmbH). Simulating the synovial fluid, non-animal stabilized hyaluronic acid was applied to the osteochondral model. In order to model the synovial membrane, a confluent hMSC layer was formed on a polycarbonate membrane. Subsequently, we applied sorted CD15<sup>+</sup> cells to the synovial membrane and cytokines (e.g. TNF, MIF, IL6) into the synovial fluid, analyzed using qPCR, multiplex immunoassay and flow cytometry. <h3>Results</h3> We developed an <i>in vitro</i> 3D bone model by successfully seeding hMSC on a β-TCP scaffold. Cells consistently adhere onto the scaffold for up to 6 weeks as observed by SEM. The analysis of cell viability via LDH detection and LIVE/DEAD staining showed no toxic effects on the cells even after 3 weeks of incubation as compared to the corresponding control. mRNA expression of bone-related genes such as <i>RUNX2</i>, <i>SPP1</i> and <i>COL1A1</i> as well as μCT analysis confirmed the osteogenic phenotype of hMSC grown in 3D. Mimicking the articular cartilage component, we verified its chondrogenic phenotype by HE and Alcian Blue staining as well as by the reduced mRNA expression of <i>COL1A1</i> and an abundant expression of <i>COL2A1</i>. Interestingly, co-cultivation of the osteogenic and chondrogenic part looks promising showing a close alignment and cell interaction between both components. Modelling the synovial membrane, we successfully and reproducibly created a confluent hMSC cell layer, with a high cell viability even after 3 weeks of incubation. In addition, we injected cytokines into the synovial fluid. In response, the simulated synovial membrane showed an upregulation of <i>IL6, HIF</i> and <i>MMP13</i> expression on mRNA level. Furthermore, under co-cultural conditions the hMSCs enhanced the survival of the PMNs. <h3>Conclusion</h3> Results from the analysis of the single components confirmed viability, integrity and morphology pointing towards an ultimately successful development of the anticipated <i>in vitro</i> 3D model. By combining the different components in a standard 96 well format, we aim to provide a mid-throughput system for preclinical drug testing as well as a valid <i>in vitro</i> human-based 3D disease model to study the pathogenesis of arthritis. <h3>Disclosure of Interests</h3> Alexandra Damerau: None declared, Annemarie Lang: None declared, Moritz Pfeiffenberger: None declared, Timo Gaber Grant/research support from: Pfizer, Frank Buttgereit: None declared