Angiopoietin-2 (Ang-2) is a key mediator of vascular disease during sepsis, and elevated plasma levels of Ang-2 are associated with organ injury scores and poor clinical outcomes. We have previously observed that biomarkers of endothelial glycocalyx (EG) damage correlate with plasma Ang-2 levels, suggesting a potential mechanistic linkage between EG injury and Ang-2 expression during states of systemic inflammation. However, the cell signaling mechanisms regulating Ang-2 expression following EG damage are unknown. In the current study, we determined the temporal associations between plasma heparan sulfate (HS) levels as a marker of EG erosion and plasma Ang-2 levels in children with sepsis and in mouse models of sepsis. Second, we evaluated the role of shear stress–mediated 5′-adenosine monophosphate–activated protein kinase (AMPK) signaling in Ang-2 expression following enzymatic HS cleavage from the surface of human primary lung microvascular endothelial cells (HLMVECs). We found that plasma HS levels peaked before plasma Ang-2 levels in children and mice with sepsis. Further, we discovered that impaired AMPK signaling contributed to increased Ang-2 expression following HS cleavage from flow-conditioned HLMVECs, establishing a paradigm by which Ang-2 may be upregulated during sepsis.

The mortality of patients suffering from acute myocardial infarction is linearly related to the infarct size. As regeneration of cardiomyocytes from cardiac progenitor cells is minimal in the mammalian adult heart, we have explored a new therapeutic approach, which leverages the capacity of nanomaterials to release chemicals over time to promote myocardial protection and infarct size reduction. Initial screening identified 2 chemicals, FGF1 and CHIR99021 (a Wnt1 agonist/GSK-3β antagonist), which synergistically enhance cardiomyocyte cell cycle in vitro. Poly-lactic-co-glycolic acid nanoparticles (NPs) formulated with CHIR99021 and FGF1 (CHIR + FGF1-NPs) provided an effective slow-release system for up to 4 weeks. Intramyocardial injection of CHIR + FGF1-NPs enabled myocardial protection via reducing infarct size by 20%–30% in mouse or pig models of postinfarction left ventricular (LV) remodeling. This LV structural improvement was accompanied by preservation of cardiac contractile function. Further investigation revealed that CHIR + FGF1-NPs resulted in a reduction of cardiomyocyte apoptosis and increase of angiogenesis. Thus, using a combination of chemicals and an NP-based prolonged-release system that works synergistically, this study demonstrates a potentially novel therapy for LV infarct size reduction in hearts with acute myocardial infarction.

Free from the limitations posed by exogenous scaffolds or extracellular matrix-based materials, scaffold-free engineered tissues have immense clinical potential. Biomaterials may produce adverse responses, interfere with cell–cell interaction, or affect the extracellular matrix integrity of cells. The scaffold-free Kenzan method can generate complex tissues using spheroids on an array of needles but could be inefficient in terms of time, as it moves and places only a single spheroid at a time. We aimed to design and construct a novel scaffold-free bioprinter that can print an entire layer of spheroids at once, effectively reducing the printing time. The bioprinter was designed using computer-aided design software and constructed from machined, 3D printed, and commercially available parts. The printing efficiency and the operating precision were examined using Zirconia and alginate beads, which mimic spheroids. In less than a minute, the printer could efficiently pick and transfer the beads to the printing surface and assemble them onto the 4 × 4 needles. The average overlap coefficient between layers was measured and found to be 0.997. As a proof of concept using human induced pluripotent stem cell-derived spheroids, we confirmed the ability of the bioprinter to place cellular spheroids onto the needles efficiently to print an entire layer of tissue. This novel layer-by-layer, scaffold-free bioprinter is efficient and precise in operation and can be easily scaled to print large tissues.

Human cardiac-muscle patches (hCMPs) constructed from induced pluripotent stem cells derived cardiomyocytes (iCMs) can replicate the genetics of individual patients, and consequently be used for drug testing, disease modeling, and therapeutic applications. However, conventional hCMPs are relatively thin and contain iCMs with fetal cardiomyocyte structure and function. Here, we used our layer-by-layer (lbl) fabrication to construct thicker (>2.1 mm), triple-layered hCMPs, and then evaluated iCM maturity after ten days of standard culture (Control), static stretching (Stretched), or stretching with electrical stimulation at 15 or 22 V (Stretched+15V or Stretched+22V). Assessments of stained hCMPs suggested that expression and alignment of contractile proteins was greater in Stretched+22V, whereas quantification of mRNA abundance and protein expression indicated the Stretched+22V enhanced biomolecular maturation. Transmission electron microscope images indicated that stretching and electrical stimulation were associated with increases in development of Z-lines and gap junctions, and sarcomeres were significantly longer following any of the maturation protocols.