The goal of the present studies was to investigate the role of changes in hydrogen sulfide (H 2 S) homeostasis in the pathogenesis of hyperglycemic endothelial dysfunction. Exposure of bEnd3 microvascular endothelial cells to elevated extracellular glucose (in vitro "hyperglycemia") induced the mitochondrial formation of reactive oxygen species (ROS), which resulted in an increased consumption of endogenous and exogenous H 2 S. Replacement of H 2 S or overexpression of the H 2 S-producing enzyme cystathionine-γ-lyase (CSE) attenuated the hyperglycemia-induced enhancement of ROS formation, attenuated nuclear DNA injury, reduced the activation of the nuclear enzyme poly(ADP-ribose) polymerase, and improved cellular viability. In vitro hyperglycemia resulted in a switch from oxidative phosphorylation to glycolysis, an effect that was partially corrected by H 2 S supplementation. Exposure of isolated vascular rings to high glucose in vitro induced an impairment of endothelium-dependent relaxations, which was prevented by CSE overexpression or H 2 S supplementation. siRNA silencing of CSE exacerbated ROS production in hyperglycemic endothelial cells. Vascular rings from CSE −/− mice exhibited an accelerated impairment of endothelium-dependent relaxations in response to in vitro hyperglycemia, compared with wild-type controls. Streptozotocininduced diabetes in rats resulted in a decrease in the circulating level of H 2 S; replacement of H 2 S protected from the development of endothelial dysfunction ex vivo. In conclusion, endogenously produced H 2 S protects against the development of hyperglycemia-induced endothelial dysfunction. We hypothesize that, in hyperglycemic endothelial cells, mitochondrial ROS production and increased H 2 S catabolism form a positive feed-forward cycle. H 2 S replacement protects against these alterations, resulting in reduced ROS formation, improved endothelial metabolic state, and maintenance of normal endothelial function.

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Increased aortic MG, AGE formation and oxidative stress were associated with blood pressure increase in SHR, which may cause endothelial dysfunction and altered vascular reactivity.

Three-dimensional (3D) bioprinting of hybrid constructs is a promising biofabrication method for cartilage tissue engineering because a synthetic polymer framework and cell-impregnated hydrogel provide structural and biological features of cartilage, respectively. During bioprinting, impregnated cells may be subjected to high temperatures (caused by the adjacent melted polymer) and process-induced mechanical forces, potentially compromising cell function. This study addresses these biofabrication issues, evaluating the heat distribution of printed polycaprolactone (PCL) strands and the rheological property and structural stability of alginate hydrogels at various temperatures and concentrations. The biocompatibility of parameters from these studies was tested by culturing 3D hybrid constructs bioprinted with primary cells from embryonic chick cartilage. During initial two-dimensional culture expansion of these primary cells, two morphologically and molecularly distinct cell populations ("rounded" and "fibroblastic") were isolated. The biological performance of each population was evaluated in 3D hybrid constructs separately. The cell viability, proliferation, and cartilage differentiation were observed at high levels in hybrid constructs of both cell populations, confirming the validity of these 3D bioprinting parameters for effective cartilage tissue engineering. Statistically significant performance variations were observed, however, between the rounded and fibroblastic cell populations. Molecular and morphological data support the notion that such performance differences may be attributed to the relative differentiation state of rounded versus fibroblastic cells (i.e., differentiated chondrocytes vs. chondroprogenitors, respectively), which is a relevant issue for cell-based tissue engineering strategies. Taken together, our study demonstrates that bioprinting 3D hybrid constructs of PCL and cell-impregnated alginate hydrogel is a promising approach for cartilage tissue engineering.

Calcified cartilage regeneration plays an important role in successful osteochondral repair, since it provides a biological and mechanical transition from the unmineralized cartilage at the articulating surface to the underlying mineralized bone. To biomimic native calcified cartilage in engineered constructs, here we test the hypothesis that hydroxyapatite (HAP) stimulates chondrocytes to secrete the characteristic matrix of calcified cartilage. Sodium citrate (SC) was added as a dispersant of HAP within alginate (ALG), and homogeneous dispersal of HAP within ALG hydrogel was confirmed using sedimentation tests, electron microscopy, and energy dispersive spectroscopy. To examine the biological performance of ALG/HAP composites, chondrocyte survival and proliferation, extracellular matrix production, and mineralization potential were evaluated in the presence or absence of the HAP phase. Chondrocytes in ALG/HAP constructs survived well and proliferated, but also expressed higher levels of calcified cartilage markers compared to controls, including Collagen type X secretion, alkaline phosphatase (ALP) activity, and mineral deposition. Compared to controls, ALG/HAP constructs also showed an elevated level of mineralized matrix in vivo when implanted subcutaneously in mice. The printability of ALG/HAP composite hydrogel precursors was verified by 3D printing of ALG/HAP hydrogel scaffolds with a porous structure. In summary, these results confirm the hypothesis that HAP in ALG hydrogel stimulates chondrocytes to secrete calcified matrix in vitro and in vivo and reveal that ALG/HAP composites have the potential for 3D bioprinting and osteochondral regeneration.