Differential Regulation of Human Bone Marrow Mesenchymal Stromal Cell Chondrogenesis by Hypoxia Inducible Factor-1α Hydroxylase Inhibitors.

The transcriptional profile induced by hypoxia plays important roles in the chondrogenic differentiation of marrow stromal/stem cells (MSC) and is mediated by the hypoxia inducible factor (HIF) complex. However, various compounds can also stabilize HIF's oxygen-responsive element, HIF-1α, at normoxia and mimic many hypoxia-induced cellular responses. Such compounds may prove efficacious in cartilage tissue engineering, where microenvironmental cues may mediate functional tissue formation. Here, we investigated three HIF-stabilizing compounds, which each have distinct mechanisms of action, to understand how they differentially influenced the chondrogenesis of human bone marrow-derived MSC (hBM-MSC) in vitro. hBM-MSCs were chondrogenically-induced in transforming growth factor-ß3-containing media in the presence of HIF-stabilizing compounds. HIF-1α stabilization was assessed by HIF-1α immunofluorescence staining, expression of HIF target and articular chondrocyte specific genes by quantitative polymerase chain reaction, and cartilage-like extracellular matrix production by immunofluorescence and histochemical staining. We demonstrate that all three compounds induced similar levels of HIF-1α nuclear localization. However, while the 2-oxoglutarate analog dimethyloxalylglycine (DMOG) promoted upregulation of a selection of HIF target genes, desferrioxamine (DFX) and cobalt chloride (CoCl2 ), compounds that chelate or compete with divalent iron (Fe2+ ), respectively, did not. Moreover, DMOG induced a more chondrogenic transcriptional profile, which was abolished by Acriflavine, an inhibitor of HIF-1α-HIF-ß binding, while the chondrogenic effects of DFX and CoCl2 were more limited. Together, these data suggest that HIF-1α function during hBM-MSC chondrogenesis may be regulated by mechanisms with a greater dependence on 2-oxoglutarate than Fe2+ availability. These results may have important implications for understanding cartilage disease and developing targeted therapies for cartilage repair. Stem Cells 2018;36:1380-1392.

Hypoxia impacts human MSC response to substrate stiffness during chondrogenic differentiation.

Tissue engineering strategies often aim to direct tissue formation by mimicking conditions progenitor cells experience within native tissues. For example, to create cartilage in vitro, researchers often aim to replicate the biochemical and mechanical milieu cells experience during cartilage formation in the developing limb bud. This includes stimulating progenitors with TGF-ß1/3, culturing under hypoxic conditions, and regulating mechanosensory pathways using biomaterials that control substrate stiffness and/or cell shape. However, as progenitors differentiate down the chondrogenic lineage, the pathways that regulate their responses to mechanotransduction, hypoxia and TGF-ß may not act independently, but rather also impact one another, influencing overall cell response. Here, to better understand hypoxia's influence on mechanoregulatory-mediated chondrogenesis, we cultured human marrow stromal/mesenchymal stem cells (hMSC) on soft (0.167 kPa) or stiff (49.6 kPa) polyacrylamide hydrogels in chondrogenic medium containing TGF-ß3. We then compared cell morphology, phosphorylated myosin light chain 2 staining, and chondrogenic gene expression under normoxic and hypoxic conditions, in the presence and absence of pharmacological inhibition of cytoskeletal tension. We show that on soft compared to stiff substrates, hypoxia prompts hMSC to adopt more spread morphologies, assemble in compact mesenchymal condensation-like colonies, and upregulate NCAM expression, and that inhibition of cytoskeletal tension negates hypoxia-mediated upregulation of molecular markers of chondrogenesis, including COL2A1 and SOX9. Taken together, our findings support a role for hypoxia in regulating hMSC morphology, cytoskeletal tension and chondrogenesis, and that hypoxia's effects are modulated, at least in part, by mechanosensitive pathways. Our insights into how hypoxia impacts mechanoregulation of chondrogenesis in hMSC may improve strategies to develop tissue engineered cartilage. STATEMENT OF SIGNIFICANCE: Cartilage tissue engineering strategies often aim to drive progenitor cell differentiation by replicating the local environment of the native tissue, including by regulating oxygen concentration and mechanical stiffness. However, the pathways that regulate cellular responses to mechanotransduction and hypoxia may not act independently, but rather also impact one another. Here, we show that on soft, but not stiff surfaces, hypoxia impacts human MSC (hMSC) morphology and colony formation, and inhibition of cytoskeletal tension negates the hypoxia-mediated upregulation of molecular markers of chondrogenesis. These observations suggest that hypoxia's effects during hMSC chondrogenesis are modulated, at least in part, by mechanosensitive pathways, and may impact strategies to develop scaffolds for cartilage tissue engineering, as hypoxia's chondrogenic effects may be enhanced on soft materials.

Exploiting Advanced Hydrogel Technologies to Address Key Challenges in Regenerative Medicine.

Regenerative medicine aims to tackle a panoply of challenges from repairing focal damage to articular cartilage to preventing pathological tissue remodeling after myocardial infarction. Hydrogels are water-swollen networks formed from synthetic or naturally derived polymers and are emerging as important tools to address these challenges. Recent advances in hydrogel chemistries are enabling researchers to create hydrogels that can act as 3D ex vivo tissue models, allowing them to explore fundamental questions in cell biology by replicating tissues' dynamic and nonlinear physical properties. Enabled by cutting edge techniques such as 3D bioprinting, cell-laden hydrogels are also being developed with highly controlled tissue-specific architectures, vasculature, and biological functions that together can direct tissue repair. Moreover, advanced in situ forming and acellular hydrogels are increasingly finding use as delivery vehicles for bioactive compounds and in mediating host cell response. Here, advances in the design and fabrication of hydrogels for regenerative medicine are reviewed. It is also addressed how controlled chemistries are allowing for precise engineering of spatial and time-dependent properties in hydrogels with a look to how these materials will eventually translate to clinical applications.