Carmine Gentile

Bio: Carmine Gentile is an academic researcher from University of Technology, Sydney. The author has contributed to research in topics: Stem cell & Medicine. The author has an hindex of 15, co-authored 38 publications receiving 1654 citations. Previous affiliations of Carmine Gentile include Sewanee: The University of the South & Harvard University.

Organ Printing: Tissue Spheroids as Building Blocks

Abstract: Organ printing can be defined as layer-by-layer additive robotic biofabrication of three-dimensional functional living macrotissues and organ constructs using tissue spheroids as building blocks. The microtissues and tissue spheroids are living materials with certain measurable, evolving and potentially controllable composition, material and biological properties. Closely placed tissue spheroids undergo tissue fusion - a process that represents a fundamental biological and biophysical principle of developmental biology-inspired directed tissue self-assembly. It is possible to engineer small segments of an intraorgan branched vascular tree by using solid and lumenized vascular tissue spheroids. Organ printing could dramatically enhance and transform the field of tissue engineering by enabling large-scale industrial robotic biofabrication of living human organ constructs with "built-in" perfusable intraorgan branched vascular tree. Thus, organ printing is a new emerging enabling technology paradigm which represents a developmental biology-inspired alternative to classic biodegradable solid scaffold-based approaches in tissue engineering.

Towards organ printing: engineering an intra-organ branched vascular tree

Abstract: Importance of the field: Effective vascularization of thick three-dimensional engineered tissue constructs is a problem in tissue engineering. As in native organs, a tissue-engineered intra-organ vascular tree must be comprised of a network of hierarchically branched vascular segments. Despite this requirement, current tissue-engineering efforts are still focused predominantly on engineering either large-diameter macrovessels or microvascular networks.Areas covered in this review: We present the emerging concept of organ printing or robotic additive biofabrication of an intra-organ branched vascular tree, based on the ability of vascular tissue spheroids to undergo self-assembly.What the reader will gain: The feasibility and challenges of this robotic biofabrication approach to intra-organ vascularization for tissue engineering based on organ-printing technology using self-assembling vascular tissue spheroids including clinically relevantly vascular cell sources are analyzed.Take home message: It is not p...

Cardiac spheroids as promising in vitro models to study the human heart microenvironment.

Abstract: Three-dimensional in vitro cell systems are a promising alternative to animals to study cardiac biology and disease. We have generated three-dimensional in vitro models of the human heart (“cardiac spheroids”, CSs) by co-culturing human primary or iPSC-derived cardiomyocytes, endothelial cells and fibroblasts at ratios approximating those present in vivo. The cellular organisation, extracellular matrix and microvascular network mimic human heart tissue. These spheroids have been employed to investigate the dose-limiting cardiotoxicity of the common anti-cancer drug doxorubicin. Viability/cytotoxicity assays indicate dose-dependent cytotoxic effects, which are inhibited by the nitric oxide synthase (NOS) inhibitor L-NIO, and genetic inhibition of endothelial NOS, implicating peroxynitrous acid as a key damaging agent. These data indicate that CSs mimic important features of human heart morphology, biochemistry and pharmacology in vitro, offering a promising alternative to animals and standard cell cultures with regard to mechanistic insights and prediction of toxic effects in human heart tissue.

Fusion of uniluminal vascular spheroids: a model for assembly of blood vessels

Abstract: We evaluated the self-assembly properties of uniluminal vascular spheroids having outer layers of vascular smooth muscle cells and a contiguous inner layer of endothelial cells lining a central lumen. We showed that while pairs of uniluminal vascular spheroids suspended in culture medium fused to form a larger diameter spheroidal structure, spheroids in collagen hydrogels formed elongated structures. These findings highlight the potential use of uniluminal vascular spheroids as modules to engineer blood vessels. We also demonstrate that uniluminal vascular spheroid fusion conforms to models describing the coalescence of liquid drops. Furthermore, the fusion of uniluminal vascular spheroids in vitro closely resembled the in vivo process by which the descending aorta forms from the fusion of the paired dorsal aortae during embryonic development. Together, the findings indicate that tissue liquidity underlies uniluminal vascular spheroid fusion and that in vivo anastomosis of blood vessels may involve a similar mechanism.

Glutathionylation mediates angiotensin II-induced eNOS uncoupling, amplifying NADPH oxidase-dependent endothelial dysfunction.

Abstract: Background Glutathionylation of endothelial nitric oxide synthase (eNOS) “uncouples” the enzyme, switching its function from nitric oxide (NO) to O2•− generation. We examined whether this reversible redox modification plays a role in angiotensin II (Ang II)-induced endothelial dysfunction. Methods and Results Ang II increased eNOS glutathionylation in cultured human umbilical vein endothelial cells (HUVECs), rabbit aorta, and human arteries in vitro. This was associated with decreased NO bioavailability and eNOS activity as well as increased O2•− generation. Ang II-induced decrease in eNOS activity was mediated by glutathionylation, as shown by restoration of function by glutaredoxin-1. Moreover, Ang II-induced increase in O2•− and decrease in NO were abolished in HUVECs transiently transfected, with mutant eNOS rendered resistant to glutathionylation. Ang II effects were nicotinamide adenine dinucleotide phosphate (NADPH) oxidase dependent because preincubation with gp 91ds-tat, an inhibitor of NADPH oxidase, abolished the increase in eNOS glutathionylation and loss of eNOS activity. Functional significance of glutathionylation in intact vessels was supported by Ang II-induced impairment of endothelium-dependent vasorelaxation that was abolished by the disulfide reducing agent, dithiothreitol. Furthermore, attenuation of Ang II signaling in vivo by administration of an angiotensin converting enzyme (ACE) inhibitor reduced eNOS glutathionylation, increased NO, diminished O2•−, improved endothelium-dependent vasorelaxation and reduced blood pressure. Conclusions Uncoupling of eNOS by glutathionylation is a key mediator of Ang II-induced endothelial dysfunction, and its reversal is a mechanism for cardiovascular protection by ACE inhibition. We suggest that Ang II-induced O2•− generation in endothelial cells, although dependent on NADPH oxidase, is amplified by glutathionylation-dependent eNOS uncoupling.

3D bioprinting of tissues and organs

Abstract: Additive manufacturing, otherwise known as three-dimensional (3D) printing, is driving major innovations in many areas, such as engineering, manufacturing, art, education and medicine. Recent advances have enabled 3D printing of biocompatible materials, cells and supporting components into complex 3D functional living tissues. 3D bioprinting is being applied to regenerative medicine to address the need for tissues and organs suitable for transplantation. Compared with non-biological printing, 3D bioprinting involves additional complexities, such as the choice of materials, cell types, growth and differentiation factors, and technical challenges related to the sensitivities of living cells and the construction of tissues. Addressing these complexities requires the integration of technologies from the fields of engineering, biomaterials science, cell biology, physics and medicine. 3D bioprinting has already been used for the generation and transplantation of several tissues, including multilayered skin, bone, vascular grafts, tracheal splints, heart tissue and cartilaginous structures. Other applications include developing high-throughput 3D-bioprinted tissue models for research, drug discovery and toxicology.

Polymers for 3D Printing and Customized Additive Manufacturing

Abstract: Additive manufacturing (AM) alias 3D printing translates computer-aided design (CAD) virtual 3D models into physical objects. By digital slicing of CAD, 3D scan, or tomography data, AM builds objects layer by layer without the need for molds or machining. AM enables decentralized fabrication of customized objects on demand by exploiting digital information storage and retrieval via the Internet. The ongoing transition from rapid prototyping to rapid manufacturing prompts new challenges for mechanical engineers and materials scientists alike. Because polymers are by far the most utilized class of materials for AM, this Review focuses on polymer processing and the development of polymers and advanced polymer systems specifically for AM. AM techniques covered include vat photopolymerization (stereolithography), powder bed fusion (SLS), material and binder jetting (inkjet and aerosol 3D printing), sheet lamination (LOM), extrusion (FDM, 3D dispensing, 3D fiber deposition, and 3D plotting), and 3D bioprinting....

A 3D bioprinting system to produce human-scale tissue constructs with structural integrity

Abstract: A challenge for tissue engineering is producing three-dimensional (3D), vascularized cellular constructs of clinically relevant size, shape and structural integrity. We present an integrated tissue-organ printer (ITOP) that can fabricate stable, human-scale tissue constructs of any shape. Mechanical stability is achieved by printing cell-laden hydrogels together with biodegradable polymers in integrated patterns and anchored on sacrificial hydrogels. The correct shape of the tissue construct is achieved by representing clinical imaging data as a computer model of the anatomical defect and translating the model into a program that controls the motions of the printer nozzles, which dispense cells to discrete locations. The incorporation of microchannels into the tissue constructs facilitates diffusion of nutrients to printed cells, thereby overcoming the diffusion limit of 100-200 μm for cell survival in engineered tissues. We demonstrate capabilities of the ITOP by fabricating mandible and calvarial bone, cartilage and skeletal muscle. Future development of the ITOP is being directed to the production of tissues for human applications and to the building of more complex tissues and solid organs.

3D Bioprinting of Vascularized, Heterogeneous Cell‐Laden Tissue Constructs

Abstract: A new bioprinting method is reported for fabricating 3D tissue constructs replete with vasculature, multiple types of cells, and extracellular matrix. These intricate, heterogeneous structures are created by precisely co-printing multiple materials, known as bioinks, in three dimensions. These 3D micro-engineered environments open new -avenues for drug screening and fundamental studies of wound healing, angiogenesis, and stem-cell niches.