Use of biomimetic microtissue spheroids and specific growth factor supplementation to improve tenocyte differentiation and adaptation to a collagen-based scaffold in vitro.
Tenocytes represent a valuable source of cells for the purposes of tendon tissue engineering and regenerative medicine and as such, should possess a high degree of tenogenic differentiation prior to their use in vivo in order to achieve maximal efficacy. In the current report, we identify an efficient means by which to maintain differentiated tenocytes in vitro by employing the hanging drop technique in combination with defined growth media supplements. Equine tenocytes retained a more differentiated state when cultured as scaffold-free microtissue spheroids in low serum-containing medium supplemented with L-ascorbic acid 2-phosphate, insulin and transforming growth factor (TGF)-β1. This was made evident by significant increases in the expression levels of pro-tenogenic markers collagen type I (COL1A2), collagen type III (COL3A1), scleraxis (SCX) and tenomodulin (TNMD), as well as by enhanced levels of collagen type I and tenomodulin protein. Furthermore, tenocytes cultured under these conditions demonstrated a typical spindle-like morphology and when embedded in collagen gels, became highly aligned with respect to the orientation of the collagen structure following their migration out from the microtissue spheroids. Our findings therefore provide evidence to support the use of a biomimetic microtissue approach to culturing tenocytes and that in combination with the defined growth media described, can improve their differentiation status and functional repopulation of collagen matrix.
Center for Applied Biotechnology and Molecular Medicine (CABMM), University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland; Equine Department, Vetsuisse-Faculty, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland; Graduate School for Cellular and Biomedical Sciences, University of Bern, 3012 Bern, Switzerland.
Felix
F
Theiss
Center for Applied Biotechnology and Molecular Medicine (CABMM), University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland; Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, 8057 Zurich, Switzerland.
Ali
A
Mirsaidi
Cartilage Engineering and Regeneration, ETH Zurich, 8093 Zurich, Switzerland; American University of Beirut, Faculty of Engineering and Architecture, Riad El Solh, 1107 2020 Beirut, Lebanon.
Rami
R
Mhanna
Center for Applied Biotechnology and Molecular Medicine (CABMM), University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland; Equine Department, Vetsuisse-Faculty, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland; Graduate School for Cellular and Biomedical Sciences, University of Bern, 3012 Bern, Switzerland.
Jan
J
Kümmerle
Center for Applied Biotechnology and Molecular Medicine (CABMM), University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland; Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, 8057 Zurich, Switzerland.
Stephan
S
Glanz
Center for Applied Biotechnology and Molecular Medicine (CABMM), University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland; Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, 8057 Zurich, Switzerland.
Gregor
G
Bahrenberg
Center for Applied Biotechnology and Molecular Medicine (CABMM), University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
André N
AN
Tiaden
Center for Applied Biotechnology and Molecular Medicine (CABMM), University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland; Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, 8057 Zurich, Switzerland. Electronic address: peter.richards@cabmm.uzh.ch.
Peter J
PJ
Richards
D000818:Animals; D001205:Ascorbic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D032701:Biomimetics; D002454:Cell Differentiation; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry*; D003470:Culture Media / Q000378:metabolism*; D006736:Horses; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D012038:Regeneration; D018874:Spheroids, Cellular; D013710:Tendons / Q000166:cytology* / Q000502:physiology; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds / Q000737:chemistry*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism
D003470:Culture Media; D036341:Intercellular Signaling Peptides and Proteins; D053773:Transforming Growth Factor beta1; C011669:ascorbate-2-phosphate; D003094:Collagen; D001205:Ascorbic Acid
Growth factors; In vitro test; Microsphere; TGF (transforming growth factor); Tendon