MAGNETISSUE BIOREACTOR FOR TENDON TISSUE ENGINEERING: EFFECT OF CYCLIC LOADING ON CELL SURVIVAL Ekaterina A. Oleinik (1,2), Andreas H. Teuschl-Woller (2), Büsra Külekci (2), Philipp J. Thurner (1) 1. Institute of Lightweight Design and Structural Biomechanics, TU Wien, Vienna, Austria 2. Department of Life Science Engineering, UAS Technikum Wien, Vienna, Austria; Introduction Tendinopathies are becoming more prevalent, yet their exact cause is not always clear. In vitro 3D models are an important approach for studying tendinopathy; however, there is no gold standard to mimic this pathology, and further research is required to overcome current limitations. Tendon models generally rely on a combination of three critical elements: cellular components, biomaterials, and regulatory signals (mechanical stimulation, growth factors, etc.) [1]. The latter requires the determination of stimulation parameters alongside scaffold selection, characterization and optimization of stimulation to induce cellular alignment and production of proper extracellular matrix (ECM). Materials and methods Here, we employed a strain-applying bioreactor (MagneTissue) [2] to establish a tendon-like tissue model. Rat tendon-derived progenitor cells (TDPCs) at a concentration of 2 * 106 cells/mL were embedded in fibrin constructs made of 20 mg/mL fibrinogen (final conc.) and 0.625 KIU thrombin [3]. Three days after casting, rings were transferred to the bioreactor and subjected to 10% uniaxial tensile strain in two different modes: static (SL) and cyclic (CL) loading. Overall, the mechanical load was applied for 6 h per day for 6 consecutive days with 0.5 mHz frequency for CL (Fig. 1). Cell viability, alignment, distribution, and density were analyzed using immunofluorescent staining. Statistical analysis was done using GraphPad Prism 8.2.1. Results are presented as mean ± standard deviation (SD), n=4 per group. Statistical differences were evaluated using the non-parametric Kruskal- Wallis test. Differences were considered significant at p<0.05. Figure 1: Experimental set-up and working principle of the MagneTissue Bioreactor. Results Cell viability did not differ in any experimental group. Although, cell nuclei were aligned towards the axis of strain in both loaded groups (SL and CL), cell distribution throughout the scaffold varied. In both groups, cells were predominantly located on the surface, with no difference in cell number in this area. However, there was a significant increase in cell number inside the construct when the cyclic load was applied compared to the non-loaded group (Fig. 2, left). Visual analysis of DAPI-stained sections confirmed fewer fragmented nuclei within the construct in the CL group compared to both SL and non-loaded groups (Fig. 2, right). Collagen I production was limited to a depth of ~300 μm from the surface (data not shown). Figure 2: DAPI-stained longitudinal slices of fibrin constructs. Images demonstrate cell localization and fragmented nuclei (pointed by arrows). Discussion Limited extracellular matrix remodeling, differences in cell number on the surface vs inside and the presence of fragmented nuclei may indicate oxygen and nutrition deprivation within the constructs and a subsequent necrotic core formation [4]. Cyclic loading appears to improve media supply, resulting in a higher number of cells inside and fewer fragmented, possibly apoptotic, nuclei. In ongoing experiments, we further assess cell proliferation, migration, the level of apoptosis, and its underlying mechanisms using TUNEL assay, Western blot and PCR analyses to optimize the cycling loading protocol to improve our tendon model. References 1. Akter F., Tissue Engineering Made Easy, 3-16, 2016. 2. Heher P. et al, Acta Biomater, 24:251-65, 2015. 3. Tomasch J. et al, Front Bioeng Biotech, 10: 836520, 2022. 4. LaBonia G.J. et al, Proteomics, 16(11-12):1814-2, 2016. Acknowledgements We want to thank Prof. Dr. Andreas Traweger (PMU) for his vital input and helpfull discussions and Dr. David Hercher (LBI) for providing tendon tissues. Funding by the Austrian Science Fund FWF doc.funds.connect doctoral college Maturetissue (DFH-28) is gratefully acknowledged.