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.

The effects of dynamic loading on the intervertebral disc

“…Furthermore, the high volumetric stress environment of the IVD could direct MSC differentiation towards a chondrogenic phenotype [16,94]. Hypoxia has been shown to impair adipogenesis and osteogenesis of human MSCs [38], and as a potent chondrogenesis promoter than dynamic compression or hydrostatic pressure in the presence of transforming growth factor b (TGF-b) [7,74,77,105]. Nevertheless, low oxygen tension and hydrostatic pressure alone without TGF-b could not initiate chondrogenesis of MSC [105].…”

Device and application development for haematopoietic stem and progenitor cell (HSPC) and mesenchymal stromal cell (MSC) 3D spheroid cultures

“…Similar to previous studies [337,342,351,353], MSC tri--lineage was inducible in 3D spheroids using the microwell platform. …”

“…Microtissues are appealing building blocks in tissue engineering applications [334,341,351,395,409], including the manufacture of cartilage microtissues from either mesenchymal stem/stromal cells (MSC) [337] or articular chondrocytes [395]. The fundamental motivation for using a microwell platform to manufacture cartilage microtissues is its capacity to reproducibly generate hundreds of uniform tissue building blocks.…”

“…A number of studies have reported that culture of MSC in 3D spheroids, rather than in 2D adherent culture, resulted in characteristics that may be useful to tissue engineering applications and regenerative medicine. These characteristics include the maintenance of differentiation potential toward the mesodermal lineages (osteogenic, adipogenic [325,335,336], and chondrogenic [337]), which could serve as tissue building blocks, and the increased secretion of proangiogenic factors such as vascular endothelial growth factor (VEGF) [327] and anti--inflammatory molecules such as tumour necrosis factor--α (TNF--α)--stimulated gene/protein--6 (TSG--6) [328, 338--340].…”

In Vitro Assessment of Migratory Behavior of Two Cell Populations in a Simple Multichannel Microdevice

“…Ethical approval for this study was granted through the Mater Health Services Human Research Ethics Committee and the Queensland University of Technology in accordance with the Australian National Health and Medical Research Council's Statement on Ethical Conduct in Research Involving Humans. Samples were diluted with phosphate-buffered saline (PBS) and mononuclear cells were enriched via density gradient centrifugation as described previously [23,24]. MSC were expanded in low-glucose DMEM (Gibco) containing 10% FBS and 1% P/S, and then sub-cultured in DMEM/F12 supplemented with 5% FBS and 1% P/S for one passage prior to use in migration assays to enable cell adaption to the base medium used in the MCF-7 monocultures and planned migration assays.…”