Background/Purpose: Although arthritis is a matter of research since more than 140 years, there is currently no valid 3D model available, which is able to mimic an inflamed joint. Thus, our ultimate goal is to develop a valid human in vitro 3D joint model in order to simulate arthritis. The model will contain all involved tissue components and cell types enabling the interactions between cells by cell contacts, signaling molecules and metabolites. As an alternative experimental setup for traditional animal models, our in vitro model will enable us to study the influence and efficacy of drug treatment.

To this end, we firstly developed all single components of the joint, namely the (1) osteogenic and (2) chondrogenic part, (3) the joint space with synovial fluid and (4) the synovial membrane, separately.

Methods: The osteogenic component was synthesized by seeding human bone marrow-derived mesenchymal stromal cells (hMSC) on β-tricalcium phosphate (TCP) coated with an additional hMSC monolayer cell-sheet and cultured for a total of 6 weeks. Survival, adhesion and structural integrity of the cells were evaluated by Scanning Electron Microscopy (SEM), LIVE/DEAD staining and cellular release of LDH. Osteogenic differentiation was analyzed both by µCT for mineralization and on gene expression level using qPCR. To mimic the chondrogenic part, a scaffold-free 3D cartilage construct was generated by chondrogenic differentiation of hMSC with intermittent mechanical stimulation. Constructs were analyzed by histology and qPCR. Non-animal stabilized hyaluronic acid was used to simulate the synovial fluid component. In order to model the synovial membrane, hMSC were differentiated towards the fibroblast lineage and then a confluent layer was formed on a polycarbonate membrane, which was visualized by histology.

Results: We developed an in vitro 3D osteogenic 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 as compared to the corresponding control. Osteogenic differentiation of hMSC grown in 3D was verified demonstrating an increase in mineralized bone volume and the induction of bone-related gene expression (RUNX2, SPP1 and COL1A1) as compared to the corresponding control. Chondrogenic phenotype was verified 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 the osteogenic and chondrogenic part for up to 3 weeks demonstrated successful colonization, connectivity and initial calcification implying a functional transitional bridging area. Modelling the synovial membrane, we successfully and reproducibly created a confluent monolayer of hMSC, which is easily transferable to the model.

Conclusion: In summary, we confirmed and validated in a standardized manner phenotypic integrity and stability of each single component. To finalize the development of healthy joint model we will combine the established parts to provide suitable 3D joint model that enables us to study the efficacy of drug treatment in vitro.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/simulating-the-pathogenesis-of-arthritis-in-vitro-by-developing-a-human-based-multicomponent-3d-joint-model/