title: Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures.
abstract: Chondrogenesis of mesenchymal stem cells (MSCs) is typically induced when they are condensed into a single aggregate and exposed to transforming growth factor-beta (TGF-beta). Hypoxia, like aggregation and TGF-beta delivery, may be crucial for complete chondrogenesis. However, the pellet dimensions and associated self-induced oxygen gradients of current chondrogenic methods may limit the effectiveness of in vitro differentiation and subsequent therapeutic uses. Here we describe the use of embryoid body-forming technology to produce microscopic aggregates of human bone marrow MSCs (BM-MSCs) for chondrogenesis. The use of micropellets reduces the formation of gradients within the aggregates, resulting in a more homogeneous and controlled microenvironment. These micropellet cultures (approximately 170 cells/micropellet) as well as conventional pellet cultures (approximately 2 x 10(5) cells/pellet) were chondrogenically induced under 20% and 2% oxygen environments for 14 days. Compared to conventional pellets under both environments, micropellets differentiated under 2% O(2) showed significantly increased sulfated glycosaminoglycan (sGAG) production and more homogeneous distribution of proteoglycans and collagen II. Aggrecan and collagen II gene expressions were increased in pellet cultures differentiated under 2% O(2) relative to 20% O(2) pellets but 2% O(2) micropellets showed even greater increases in these genes, as well as increased SOX9. These results suggest a more advanced stage of chondrogenesis in the micropellets accompanied by more homogeneous differentiation. Thus, we present a new method for enhancing MSC chondrogenesis that reveals a unique relationship between oxygen tension and aggregate size. The inherent advantages of chondrogenic micropellets over a single macroscopic aggregate should allow for easy integration with a variety of cartilage engineering strategies.
authors.affiliation: Tissue Engineering & Microfluidics Laboratory, Australian Institute for Bioengineering & Nanotechnology, The University of Queensland, Brisbane, Australia.
authors.forename: Brandon D
authors.initials: BD
authors.lastname: Markway
authors.forename: Guak-Kim
authors.initials: GK
authors.lastname: Tan
authors.forename: Gary
authors.initials: G
authors.lastname: Brooke
authors.forename: James E
authors.initials: JE
authors.lastname: Hudson
authors.forename: Justin J
authors.initials: JJ
authors.lastname: Cooper-White
authors.forename: Michael R
authors.initials: MR
authors.lastname: Doran
mesh_terms: D053674:Aggrecans / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D002449:Cell Aggregation / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology*; D019070:Cell Lineage / Q000502:physiology; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D020219:Chondrogenesis / Q000502:physiology*; D024043:Collagen Type II / Q000378:metabolism; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D000860:Hypoxia / Q000378:metabolism* / Q000503:physiopathology; D045164:Mesenchymal Stem Cell Transplantation / Q000379:methods*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D010101:Oxygen Consumption / Q000502:physiology; D018874:Spheroids, Cellular / Q000166:cytology / Q000378:metabolism
chemical_list: D053674:Aggrecans; D024043:Collagen Type II; D006025:Glycosaminoglycans