ABSTRACT
Lithography-based three-dimensional (3D) printing technologies allow high spatial resolution that exceeds that of typical extrusion-based bioprinting approaches, allowing to better mimic the complex architecture of biological tissues. Additionally, lithographic printing via digital light processing (DLP) enables fabrication of free-form lattice and patterned structures which cannot be easily produced with other 3D printing approaches. While significant progress has been dedicated to the development of cell-laden bioinks for extrusion-based bioprinting, less attention has been directed towards the development of cyto-compatible bio-resins and their application in lithography-based biofabrication, limiting the advancement of this promising technology. In this study, we developed a new bio-resin based on methacrylated poly(vinyl alcohol) (PVA-MA), gelatin-methacryloyl (Gel-MA) and a transition metal-based visible light photoinitiator. The utilization of a visible light photo-initiating system displaying high molar absorptivity allowed the bioprinting of constructs with high resolution features, in the range of 25-50 µm. Biofunctionalization of the resin with 1 wt% Gel-MA allowed long term survival (>90%) of encapsulated cells up to 21 d, and enabled attachment and spreading of endothelial cells seeded on the printed hydrogels. Cell-laden hydrogel constructs of high resolution with complex and ordered architecture were successfully bioprinted, where the encapsulated cells remained viable, homogenously distributed and functional. Bone and cartilage tissue synthesis was confirmed by encapsulated stem cells, underlining the potential of these DLP-bioprinted hydrogels for tissue engineering and biofabrication. Overall, the PVA-MA/Gel-MA bio-resin is a promising material for biofabrication and provides important cues for the further development of lithography-based bioprinting of complex, free-form living tissue analogues.
Subject(s)
Acrylic Resins/chemistry , Bioprinting/methods , Cell Culture Techniques/methods , Tissue Scaffolds/chemistry , Cell Differentiation , Cell Survival , Cells, Cultured , Gelatin/chemistry , Humans , Hydrogels/chemistry , Light , Methacrylates/chemistry , Polyvinyl Alcohol/chemistry , Tissue Engineering/methodsABSTRACT
Activation of Rap1 by exchange protein activated by cAMP (Epac) promotes cell adhesion and actin cytoskeletal polarization. Pharmacologic activation of Epac-Rap signaling by the Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP during ischemia-reperfusion (IR) injury reduces renal failure and application of 8-pCPT-2'-O-Me-cAMP promotes renal cell survival during exposure to the nephrotoxicant cisplatin. Here, we found that activation of Epac by 8-pCPT-2'-O-Me-cAMP reduced production of reactive oxygen species during reoxygenation after hypoxia by decreasing mitochondrial superoxide production. Epac activation prevented disruption of tubular morphology during diethyl maleate-induced oxidative stress in an organotypic three-dimensional culture assay. In vivo renal targeting of 8-pCPT-2'-O-Me-cAMP to proximal tubules using a kidney-selective drug carrier approach resulted in prolonged activation of Rap1 compared with nonconjugated 8-pCPT-2'-O-Me-cAMP. Activation of Epac reduced antioxidant signaling during IR injury and prevented tubular epithelial injury, apoptosis, and renal failure. Our data suggest that Epac1 decreases reactive oxygen species production by preventing mitochondrial superoxide formation during IR injury, thus limiting the degree of oxidative stress. These findings indicate a new role for activation of Epac as a therapeutic application in renal injury associated with oxidative stress.
Subject(s)
Guanine Nucleotide Exchange Factors/physiology , Kidney Tubules, Proximal/metabolism , Oxidative Stress , Urothelium/metabolism , Animals , Cyclic AMP/analogs & derivatives , Cyclic AMP/pharmacology , Guanine Nucleotide Exchange Factors/drug effects , Kidney Tubules, Proximal/drug effects , Male , Mice , Mice, Inbred C57BL , Signal Transduction , Urothelium/drug effectsABSTRACT
The development of a macromolecular conjugate of a multitargeted tyrosine kinase inhibitor is described that can be used for renal-specific delivery into proximal tubular cells. A novel sunitinib analogue, that is, 17864, is conjugated to a NH(2) -PAMAM-G3 dendrimer via the platinum (II)-based Universal Linkage System (ULS™). The activity of 17864 is retained after coordination to the ULS linker alone or when coupled to NH(2) -PAMAM-G3. 17864-UlS-NH(2) -PAMAM-G3 is non-toxic to proximal tubular cells in vitro. After intravenous administration to mice, 17864-UlS-NH(2) -PAMAM-G3 rapidly and efficiently accumulates in the kidneys. These results are encouraging for future studies focusing on the development of novel therapeutics for the treatment of renal diseases.
