dc.description.abstract
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.
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