Background/Purpose:

Our goal is to develop an in vitro 3D joint model to simulate the immune mediated pathogenesis of arthritides in order to present an alternative experimental setup for traditional animal models. There are currently no appropriate in vitro models capable of simulating an arthritic joint in such a comprehensive way that all relevant humoral and cellular factors as well as the different tissue types are involved. Therefore, we work to establish an in vitro 3D healthy joint model, followed by the simulation of an arthritic joint. The in vitro 3D joint model is planned to consist of an (1) osteogenic part and (2) a chondrogenic part, (3) the joint space with synovial fluid, and (4) the synovial membrane.

Development of the first two components of our in vitro 3D joint model, namely (1) the osteogenic and (2) the chondrogenic part.

Methods:

We used β-tricalcium phosphate (TCP) with 60% porosity as the mineral part of our osteogenic model. This substrate was populated with human bone marrow derived mesenchymal stromal cells (hMSC) predifferentiated towards the osteoblastic lineage and then cultured for up to 21 days under normoxic conditions (37 °C, 18% O₂). The adhesion of the cells and their structural integrity were evaluated by Scanning Electron Microscopy and Laser Scanning Microscopy. To confirm cell attachment and biocompatibility of β-TCP particles, cellular release of LDH (after 1 d) was assessed, and LIVE/DEAD staining was applied (1, 7, 14, 21 d). Osteogenic differentiation was verified on gene expression level using qRT-PCR. The chondrogenic model, a scaffold-free 3D cartilage construct (fzmb GmbH), was generated using native hMSC. Chondrogenic differentiation was performed under hypoxia (37 °C, 1% O₂) with intermittent mechanical stimulation and analyzed by histology (HE staining) and immunohistology (Col1a1, Col2a1, Alcian Blue).

Results:

We have been able to successfully develop an in vitro 3D bone model by seeding predifferentiated hMSC (after 24-hour preincubation with osteogenic medium) on a β-TCP scaffold. The analysis of cell viability via LDH detection did not show any toxic effects during cultivation of the cells seeded. Histological and immunofluorescence analyses demonstrated good cell attachment, cell integrity and metabolic activity of the β-TCP scaffold until day 21, demonstrating the good suitability of both the scaffold and the cultivation method. mRNA expression of bone-related genes such as RUNX2, SPP1 and COL1A1 confirmed phenotypic changes as induced during osteogenic differentiation. The development of a cartilage phenotype from the accordingly differentiated hMSC was confirmed by HE and Alcian Blue staining as well as by Col1a1 and Col2a1 expression. Histological analyses performed after 21 d of co-cultivation showed successful colonization, connectivity and initial calcification implying a functional transitional bridging area.

Conclusion: These initial results from our in vitro 3D models confirm good cell vitality indicating successful progression of developing our model. This 3D multi-component joint model should enable us to simulate arthritis and to study the efficacy of drug treatment in vitro.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/development-of-a-multi-component-3d-arthritic-joint-model/