Abstract
BackgroundLowering oxygen from atmospheric level (hyperoxia) to the physiological level (physioxia) of articular cartilage promotes mesenchymal stem cell (MSC) chondrogenesis. However, the literature is equivocal regarding the benefits of physioxic culture on preventing hypertrophy of MSC-derived chondrocytes. Articular cartilage progenitors (ACPs) undergo chondrogenic differentiation with reduced hypertrophy marker expression in hyperoxia but have not been studied in physioxia. This study sought to delineate the effects of physioxic culture on both cell types undergoing chondrogenesis.MethodsMSCs were isolated from human bone marrow aspirates and ACP clones were isolated from healthy human cartilage. Cells were differentiated in pellet culture in physioxia (2 % oxygen) or hyperoxia (20 % oxygen) over 14 days. Chondrogenesis was characterized by biochemical assays and gene and protein expression analysis.ResultsMSC preparations and ACP clones of high intrinsic chondrogenicity (termed high-GAG) produced abundant matrix in hyperoxia and physioxia. Poorly chondrogenic cells (low-GAG) demonstrated a significant fold-change matrix increase in physioxia. Both high-GAG and low-GAG groups of MSCs and ACPs significantly upregulated chondrogenic genes; however, only high-GAG groups had a concomitant decrease in hypertrophy-related genes. High-GAG MSCs upregulated many common hypoxia-responsive genes in physioxia while low-GAG cells downregulated most of these genes. In physioxia, high-GAG MSCs and ACPs produced comparable type II collagen but less type I collagen than those in hyperoxia. Type X collagen was detectable in some ACP pellets in hyperoxia but reduced or absent in physioxia. In contrast, type X collagen was detectable in all MSC preparations in hyperoxia and physioxia.ConclusionsMSC preparations and ACP clones had a wide range of chondrogenicity between donors. Physioxia significantly enhanced the chondrogenic potential of both ACPs and MSCs compared with hyperoxia, but the magnitude of response was inversely related to intrinsic chondrogenic potential. Discrepancies in the literature regarding MSC hypertrophy in physioxia can be explained by the use of low numbers of preparations of variable chondrogenicity. Physioxic differentiation of MSC preparations of high chondrogenicity significantly decreased hypertrophy-related genes but still produced type X collagen protein. Highly chondrogenic ACP clones had significantly lower hypertrophic gene levels, and there was little to no type X collagen protein in physioxia, emphasizing the potential advantage of these cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0419-8) contains supplementary material, which is available to authorized users.
Highlights
Lowering oxygen from atmospheric level to the physiological level of articular cartilage promotes mesenchymal stem cell (MSC) chondrogenesis
There existed a wide range of both baseline GAG content in hyperoxia and fold-change GAG content from hyperoxia to physioxia across individual populations of cells (Fig. 1a)
Each biologic replicate of MSCs and Articular cartilage progenitor (ACP) was categorized as high-GAG or low-GAG based on a threshold defined by their total GAG production in hyperoxia relative to that of pellet cultures of healthy human articular chondrocytes in the same conditions (Additional file 1: Figure S1)
Summary
Lowering oxygen from atmospheric level (hyperoxia) to the physiological level (physioxia) of articular cartilage promotes mesenchymal stem cell (MSC) chondrogenesis. Tissue engineering strategies to repair articular cartilage remain limited by our inability to differentiate cells in vitro toward the stable articular cartilage tissue phenotype, which is characterized by high type II collagen and aggrecan and low type I and type X collagen in the extracellular matrix. The physiologic oxygen tension (physioxia) within tissues in the human body is well below the atmospheric level (hyperoxia), with the highest level in the alveoli (110 mmHg) and arterial blood (100 mmHg) [3]. Oxygen-mediated mechanisms drive anabolism of the extracellular matrix molecules toward the articular cartilage phenotype, but the role of physioxia remains equivocal with regard to MSC terminal differentiation toward the hypertrophic phenotype
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