Stirred culture of cartilaginous microtissues promotes chondrogenic hypertrophy through exposure to intermittent shear stress

Niki Loverdou; Maxim Cuvelier; Gabriella Nilsson Hall; An-Sofie Christiaens; Isaak Decoene; Kristel Bernaerts; Bart Smeets; Herman Ramon; Frank P Luyten; Liesbet Geris; Ioannis Papantoniou

doi:10.1002/btm2.10468

Stirred culture of cartilaginous microtissues promotes chondrogenic hypertrophy through exposure to intermittent shear stress

Bioeng Transl Med. 2022 Dec 29;8(3):e10468. doi: 10.1002/btm2.10468. eCollection 2023 May.

Authors

Niki Loverdou^{1

2

3

4}, Maxim Cuvelier^{1

5}, Gabriella Nilsson Hall^{1

2}, An-Sofie Christiaens^{6

7}, Isaak Decoene^{1

2}, Kristel Bernaerts^{6

7}, Bart Smeets^{1

2

5}, Herman Ramon⁵, Frank P Luyten^{1

2}, Liesbet Geris^{1

2

3

4}, Ioannis Papantoniou^{1

2

8}

Affiliations

¹ Prometheus, Division of Skeletal Tissue Engineering KU Leuven Leuven Herestraat Belgium.
² Skeletal Biology & Engineering Research Centre, Department of Development & Regeneration KU Leuven Leuven Herestraat Belgium.
³ Biomechanics Research Unit GIGA-R In Silico Medicine, Université de Liege, Avenue de l'Hôpital 11-BAT 34 Liège 1 Belgium.
⁴ Biomechanics Section, KU Leuven Celestijnenlaan Leuven Belgium.
⁵ Biosystems Department MeBioS, KU Leuven Kasteelpark Arenberg Leuven Belgium.
⁶ Department of Chemical Engineering KU Leuven Celestijnenlaan Leuven Belgium.
⁷ Leuven Chem&Tech Celestijnenlaan Leuven Belgium.
⁸ Institute of Chemical Engineering Sciences, Foundation for Research and Technology-Hellas (FORTH) Stadiou St, Platani Patras Greece.

Abstract

Cartilage microtissues are promising tissue modules for bottom up biofabrication of implants leading to bone defect regeneration. Hitherto, most of the protocols for the development of these cartilaginous microtissues have been carried out in static setups, however, for achieving higher scales, dynamic process needs to be investigated. In the present study, we explored the impact of suspension culture on the cartilage microtissues in a novel stirred microbioreactor system. To study the effect of the process shear stress, experiments with three different impeller velocities were carried out. Moreover, we used mathematical modeling to estimate the magnitude of shear stress on the individual microtissues during dynamic culture. Identification of appropriate mixing intensity allowed dynamic bioreactor culture of the microtissues for up to 14 days maintaining microtissue suspension. Dynamic culture did not affect microtissue viability, although lower proliferation was observed as opposed to the statically cultured ones. However, when assessing cell differentiation, gene expression values showed significant upregulation of both Indian Hedgehog (IHH) and collagen type X (COLX), well known markers of chondrogenic hypertrophy, for the dynamically cultured microtissues. Exometabolomics analysis revealed similarly distinct metabolic profiles between static and dynamic conditions. Dynamic cultured microtissues showed a higher glycolytic profile compared with the statically cultured ones while several amino acids such as proline and aspartate exhibited significant differences. Furthermore, in vivo implantations proved that microtissues cultured in dynamic conditions are functional and able to undergo endochondral ossification. Our work demonstrated a suspension differentiation process for the production of cartilaginous microtissues, revealing that shear stress resulted to an acceleration of differentiation towards hypertrophic cartilage.

Keywords: bioreactor; cartilage microtissues; exometabolomics; mathematical modeling; shear stress.