[en] 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.
Disciplines :
Engineering, computing & technology: Multidisciplinary, general & others Biotechnology
Author, co-author :
Loverdou, Niki ; Université de Liège - ULiège > GIGA ; Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium ; Skeletal Biology & Engineering Research Centre, Department of Development & Regeneration, KU Leuven, Leuven, Belgium ; Biomechanics Section, KU Leuven, Leuven, Belgium
Cuvelier, Maxim; Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium ; Biosystems Department, MeBioS, KU Leuven, Leuven, Belgium
Nilsson Hall, Gabriella; Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium ; Skeletal Biology & Engineering Research Centre, Department of Development & Regeneration, KU Leuven, Leuven, Belgium
Christiaens, An-Sofie; Department of Chemical Engineering, KU Leuven, Leuven, Belgium ; Leuven Chem&Tech, Leuven, Belgium
Decoene, Isaak; Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium ; Skeletal Biology & Engineering Research Centre, Department of Development & Regeneration, KU Leuven, Leuven, Belgium
Bernaerts, Kristel; Department of Chemical Engineering, KU Leuven, Leuven, Belgium ; Leuven Chem&Tech, Leuven, Belgium
Smeets, Bart; Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium ; Skeletal Biology & Engineering Research Centre, Department of Development & Regeneration, KU Leuven, Leuven, Belgium ; Biosystems Department, MeBioS, KU Leuven, Leuven, Belgium
Ramon, Herman; Biosystems Department, MeBioS, KU Leuven, Leuven, Belgium
Luyten, Frank P.; Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium ; Skeletal Biology & Engineering Research Centre, Department of Development & Regeneration, KU Leuven, Leuven, Belgium
Geris, Liesbet ; Université de Liège - ULiège > Département d'aérospatiale et mécanique > Génie biomécanique ; Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium ; Skeletal Biology & Engineering Research Centre, Department of Development & Regeneration, KU Leuven, Leuven, Belgium ; Biomechanics Section, KU Leuven, Leuven, Belgium
Papantoniou, Ioannis ; Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Leuven, Belgium ; Skeletal Biology & Engineering Research Centre, Department of Development & Regeneration, KU Leuven, Leuven, Belgium ; Institute of Chemical Engineering Sciences, Foundation for Research and Technology-Hellas (FORTH), Patras, Greece
Language :
English
Title :
Stirred culture of cartilaginous microtissues promotes chondrogenic hypertrophy through exposure to intermittent shear stress
European Union's Horizon 2020, Grant/Award Number: 874837; European Research Council CoG INSITE, Grant/Award Number: 772418; Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture FNRS‐FRIA Met4Quality, Grant/Award Number: F3/5/8‐XH/NC6261FC; Fonds Wetenschappelijk Onderzoek (Fund for Scientific Research Flanders/FWO‐Vlaand), Grant/Award Numbers: G085018N, G0A4718N; Special Research Fund for KU Leuven, Grant/Award Number: C24/17/077 Funding informationKathleen Bosmans is thanked for performing in vivo experiments, Inge Van Hoven and Samuel Ribeiro Viseu for their experimental assistance. The research leading to this publication received funding from the Fonds Nationale de la Recherche Scientifique (FRS‐FNRS, FRIA mandate), the Fund for Scientific Research Flanders (FWO‐Vlaanderen G085018N and G0A4718N), the Special Research Fund of the KU Leuven (C24/17/077) and European Union's Horizon 2020 research and innovation program under grant agreements No. 772418 (European Research Council CoG INSITE). The project leading to this publication has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No. 874837. Liquid Chromatography–Mass Spectrometry (LC–MS) analysis was performed in the Metabolomics Core Facility, VIB, KU Leuven. Images were recorded on a Zeiss LSM 880—Airyscan (Cell and Tissue Imaging Cluster, CIC), Supported by Hercules AKUL/15/37_GOH1816N and FWO G.0929.15 to Pieter Vanden Berghe, KU Leuven.
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