title: Endogenous DKK1 and FRZB Regulate Chondrogenesis and Hypertrophy in Three-Dimensional Cultures of Human Chondrocytes and Human Mesenchymal Stem Cells.
abstract: Hypertrophic differentiation occurs during in vitro chondrogenesis of mesenchymal stem cells (MSCs), decreasing the quality of the cartilage construct. Previously we identified WNT pathway antagonists Dickkopf 1 homolog (DKK1) and frizzled-related protein (FRZB) as key factors in blocking hypertrophic differentiation of human MSCs (hMSCs). In this study, we investigated the role of endogenously expressed DKK1 and FRZB in chondrogenesis of hMSC and chondrocyte redifferentiation and in preventing cell hypertrophy using three relevant human cell based systems, isolated hMSCs, isolated primary human chondrocytes (hChs), and cocultures of hMSCs with hChs for which we specifically designed neutralizing nano-antibodies. We selected and tested variable domain of single chain heavy chain only antibodies (VHH) for their ability to neutralize the function of DKK1 or FRZB. In the presence of DKK1 and FRZB neutralizing VHH, glycosaminoglycan and collagen type II staining were significantly reduced in monocultured hMSCs and monocultured chondrocytes. Furthermore, in cocultures, cells in pellets showed hypertrophic differentiation. In conclusion, endogenous expression of the WNT antagonists DKK1 and FRZB is necessary for multiple steps during chondrogenesis: first DKK1 and FRZB are indispensable for the initial steps of chondrogenic differentiation of hMSCs, second they are necessary for chondrocyte redifferentiation, and finally in preventing hypertrophic differentiation of articular chondrocytes.
authors.affiliation: 1 Department of Developmental BioEngineering, Faculty of Science and Technology, MIRA-Institute for Biomedical Technology and Technical Medicine, University of Twente , Enschede, the Netherlands .
authors.forename: Leilei
authors.initials: L
authors.lastname: Zhong
authors.affiliation: 1 Department of Developmental BioEngineering, Faculty of Science and Technology, MIRA-Institute for Biomedical Technology and Technical Medicine, University of Twente , Enschede, the Netherlands .
authors.forename: Xiaobin
authors.initials: X
authors.lastname: Huang
authors.affiliation: 1 Department of Developmental BioEngineering, Faculty of Science and Technology, MIRA-Institute for Biomedical Technology and Technical Medicine, University of Twente , Enschede, the Netherlands .
authors.forename: Emilie Dooms
authors.initials: ED
authors.lastname: Rodrigues
authors.affiliation: 1 Department of Developmental BioEngineering, Faculty of Science and Technology, MIRA-Institute for Biomedical Technology and Technical Medicine, University of Twente , Enschede, the Netherlands .
authors.forename: Jeroen C H
authors.initials: JC
authors.lastname: Leijten
authors.affiliation: 2 QVQ BV , Utrecht, the Netherlands .
authors.forename: Theo
authors.initials: T
authors.lastname: Verrips
authors.affiliation: 2 QVQ BV , Utrecht, the Netherlands .
authors.forename: Mohamed
authors.initials: M
authors.lastname: El Khattabi
authors.affiliation: 1 Department of Developmental BioEngineering, Faculty of Science and Technology, MIRA-Institute for Biomedical Technology and Technical Medicine, University of Twente , Enschede, the Netherlands .
authors.forename: Marcel
authors.initials: M
authors.lastname: Karperien
authors.affiliation: 1 Department of Developmental BioEngineering, Faculty of Science and Technology, MIRA-Institute for Biomedical Technology and Technical Medicine, University of Twente , Enschede, the Netherlands .
authors.forename: Janine N
authors.initials: JN
authors.lastname: Post
mesh_terms: D057134:Antibodies, Neutralizing / Q000494:pharmacology; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D020219:Chondrogenesis / Q000187:drug effects; D018920:Coculture Techniques; D006023:Glycoproteins / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D006984:Hypertrophy; D036341:Intercellular Signaling Peptides and Proteins / Q000276:immunology / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D059630:Mesenchymal Stem Cells / Q000187:drug effects / Q000378:metabolism*; D008875:Middle Aged; D061905:Single-Domain Antibodies / Q000276:immunology; D060449:Wnt Signaling Pathway / Q000187:drug effects; D000099274:Frizzled-Related Proteins
chemical_list: D057134:Antibodies, Neutralizing; D015415:Biomarkers; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D061905:Single-Domain Antibodies; D000099274:Frizzled-Related Proteins; C501126:DKK1 protein, human
keywords: MSC; cell signaling; chondrogenesis; differentiation