Artikel
The 3D human-based in vitro arthritic joint model
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Veröffentlicht: | 8. Oktober 2019 |
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Gliederung
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Background: Our ultimate goal is to study the influence and efficacy of potential drug candidates in an experimental setting of arthritis. Therefore, we aim to develop a valid human in vitro 3D joint model mimicking features of joint inflammation by applying inflammatory conditions namely immune cells and pro-inflammatory cytokines. The in vitro 3D joint model consists of different components including an osteogenic and chondrogenic part, the joint space with synovial fluid and the synovial membrane.
Methods: Based on human bone marrow derived mesenchymal stromal cells (hMSCs), we developed the different 3D tissue components of the joint that were characterized in detail (e.g. cell vitality, morphology, structural integrity) using histological, biochemical and molecular biological methods, µCT and scanning electron microscopy. To mimic the 3D osteogenic component, we populated β-tricalcium phosphate (TCP) , while the 3D cartilage component was generated without scaffold. A confluent hMSC-layer seeded on a polycarbonate membrane on top of a cavity filled with hyaluronic acid (HA) represents the synovial membrane and the synovial fluid. To simulate the inflamed joint, we applied sorted neutrophils on the membrane and cytokines such as TNFα into the HA. The 'inflamed' scenario was analyzed by qPCR, multiplex immunoassay and flow cytometry.
Results: As a result, we confirmed the osteogenic phenotype of hMSC on the 3D-β-TCP-scaffold by expression of bone-related genes and µCT. Mimicking the cartilage component, we verified its chondrogenic phenotype by HE and Alcian Blue staining as well as by the minor expression of COL1A1 and an abundant expression of COL2A1. Co-cultivation of both components demonstrated a close alignment and cellular interaction. Modelling the synovial membrane, we successfully and reproducibly created a viable confluent hMSC-layer for up to 3 weeks of cultivation on top of the cavity filled with HA. When injecting cytokines into the HA, the hMSC-layer demonstrated an upregulation of IL6, HIF1A and MMP13 expression, while applying neutrophils on the layer enhanced their survival.
Conclusion: Results from the analysis of the single components confirmed viability, integrity and morphology pointing towards the successful development of the anticipated in vitro 3D joint model. By combining the different components in a standard 96 well format, we provide a mid-throughput system for preclinical drug testing as well as a valid human-based in vitro 3D disease model to study the pathogenesis of arthritis.