Charité-Universitätsmedizin Berlin, Free University and Humboldt University of Berlin Berlin, Germany
Alexandra Damerau1, Duc Ha Do Nguyen1, Christina Lubahn1, Thomas Leeuw2, Timo Gaber3 and Frank Buttgereit3, 1Charité-Universitätsmedizin Berlin, Berlin, Germany, 2Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany, 3Charité Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin / DRFZ Berlin, Berlin, Germany
Background/Purpose: Degenerative joint diseases such as osteoarthritis (OA) ultimately result in the breakdown of cartilage. The exact underlying mechanisms of cause and progression of OA remain fairly unclear. Therefore, developing a complex in vitro human joint model that mimic the 3D architecture and the metabolic, humoral and cellular interplay of the joint components are needed to study the course of OA pathogenesis. Beside the impact of metabolic components and 3D architecture, mechanical forces are important modulators of joint health, while aberrant forces are primary etiological factors leading to cartilage degeneration.
Here, we aimed to (i) develop a human in vitro 3D cartilage model using alternated perfused cultivation and (ii) simulate TNFα-mediated cartilage degradation. As a mechanical force we used the perfusion-mediated fluid shear stress (FSS) to mimic FSS during joint movement.
Methods: Human bone marrow-derived mesenchymal stromal cells (MSC) were used to develop in vitro 3D cartilage models. These models were maintained in a bioreactor with a perfusion cycle facilitating mechanical stimulation via FSS and daily sampling. Within the bioreactor, MSC mass cultures were subjected to FSS at 10 dyn/cm2 by medium circulation three times a day for 1.5h. We compared the perfused approach using an optimized FSS rate, cycles, and cultivation period of 18d to a non-perfused control. We measured cell viability, apoptosis, metabolic activity, chondrogenic gene expression (ACAN, COMP, COL2A1, COL1A1) and matrix metalloproteinase expression (MMP-1, -3, -13).
Results: Alternate perfused long-term cultivation at 10 dyn/cm² did not affect cell survival; instead, it reduced apoptosis compared to the non-perfused control. It did not affect oxygen consumption but reduced glucose consumption and lactate production and enhanced chondrogenic gene expression with reduced MMP13 and COMP gene expression compared to the non-perfused control. Mimicking pathophysiology of OA, we stimulated the model with 100 ng/mL TNFα for 6h under non-perfused and perfused long-term cultivation with FSS at 10 dyn/cm². Compared to untreated perfused conditions, TNFα stimulation (i) did not affect cell viability but enhanced apoptosis (demonstrating efficacy of stimulation), (ii) did not affect cell respiration and glycolysis, and (iii) enhanced COMP and MMP1 expression as markers of matrix protein turnover. Compared to TNFα-treated cells under non-perfused conditions, TNFα stimulation under perfused conditions (i) did not affect cell survival but reduced apoptosis, (ii) did not affect respiration but reduced glycolysis, and (iii) reduced the gene expression of IL6 but not TNFA, and (iv) diminished secreted IL-6 but enhanced TNFα. Furthermore, TNFα stimulation (v) reduced the gene expression of matrix degrading enzymes but (vi) enhanced anabolic chondrogenic matrix proteins.
Conclusion: In a 3D model that mimics OA, FSS as a mechanical stimulus provides a metabolic "feel-good" niche that reduces chondrocyte apoptosis, metabolic activity, and matrix metalloproteinase expression, increases matrix protein expression and protects against TNFα-mediated cartilage degradation.
Disclosures: A. Damerau, None; D. Do Nguyen, None; C. Lubahn, None; T. Leeuw, Sanofi-Aventis Deutschland GmbH; T. Gaber, None; F. Buttgereit, Horizon Therapeutics, Roche, Abbvie, AstraZeneca, Gruenenthal, Mundipharma, Pfizer.
