Reconstruction with hypopharyngo-gastrostomy after esophagus extirpation due to spontaneous esophageal perforation. / [Nyelocso-perforatio miatt végzett oesophagusexstirpatio rekonstrukciója hypopharyngogastrostoma képzésével].

Despite the significant improvement in surgical and intensive care therapy, esophageal perforation is still a severe, life-threatening condition. As the underlying causes, the accompanying disorders, the localization and the extent of the inflammation vary, the surgeon may sometimes encounter unexpected situations. A 58-year-old female developed necrotizing mediastinitis due to esophageal perforation as the result of incarcerated thoracic hernia of the stomach, therefore, we had to perform esophagus extirpation and cervical esophagostomy. During the reconstruction of the intestinal tract, we found shrinkage of the complete esophageal stump with unknown cause. The gastric sleeve was joined to the hypopharynx. Insufficiency was solved with conservative therapy. The patient regained partial swallowing ability after complex dysphagia treatment. Hyophapharyngo-gastrostomy done due to non-malignant disease is extremely rare in the literature, however, it can be a surgical technique of choice if required as in our case. It should be followed by rehabilitation done by a team, with emphasis on dysphagia treatment. Orv Hetil. 2020; 161(18): 756-760.

Advanced Cell and Tissue Biomanufacturing.

This position paper assesses state-of-the-art advanced biomanufacturing and identifies paths forward to advance this emerging field in biotechnology and biomedical engineering, including new research opportunities and translational and corporate activities. The vision for the field is to see advanced biomanufacturing emerge as a discipline in academic and industrial communities as well as a technological opportunity to spur research and industry growth. To navigate this vision, the paths to move forward and to identify major barriers were a focal point of discussions at a National Science Foundation-sponsored workshop focused on the topic. Some of the major needs include but are not limited to the integration of specific scientific and engineering disciplines and guidance from regulatory agencies, infrastructure requirements, and strategies for reliable systems integration. Some of the recommendations, major targets, and opportunities were also outlined, including some "grand challenges" to spur interest and progress in the field based on the participants at the workshop. Many of these recommendations have been expanded, materialized, and adopted by the field. For instance, the formation of an initial collaboration network in the community was established. This report provides suggestions for the opportunities and challenges to help move the field of advanced biomanufacturing forward. The field is in the early stages of effecting science and technology in biomanufacturing with a bright and important future impact evident based on the rapid scientific advances in recent years and industry progress.

Biofabrication: A Guide to Technology and Terminology.

Biofabrication holds the potential to generate constructs that more closely recapitulate the complexity and heterogeneity of tissues and organs than do currently available regenerative medicine therapies. Such constructs can be applied for tissue regeneration or as in vitro 3D models. Biofabrication is maturing and growing, and scientists with different backgrounds are joining this field, underscoring the need for unity regarding the use of terminology. We therefore believe that there is a compelling need to clarify the relationship between the different concepts, technologies, and descriptions of biofabrication that are often used interchangeably or inconsistently in the current literature. Our objective is to provide a guide to the terminology for different technologies in the field which may serve as a reference for the biofabrication community.

Self-assembly of tissue spheroids on polymeric membranes.

In this study, multicellular tissue spheroids were fabricated on polymeric membranes in order to accelerate the fusion process and tissue formation. To this purpose, tissue spheroids composed of three different cell types, myoblasts, fibroblasts and neural cells, were formed and cultured on agarose and membranes of polycaprolactone (PCL) and chitosan (CHT). Membranes prepared by a phase-inversion technique display different physicochemical, mechanical and transport properties, which can affect the fusion process. The membranes accelerated the fusion process of a pair of spheroids with respect to the inert substrate. In this process, a critical role is played by the membrane properties, especially by their mechanical characteristics and oxygen and carbon dioxide mass transfer. The rate of fusion was quantified and found to be similar for fibroblast, myoblast and neural tissue spheroids on membranes, which completed the fusion within 3 days. These spheroids underwent faster fusion and maturation on PCL membrane than on agarose, the rate of fusion being proportional to the value of oxygen and carbon dioxide permeances and elastic characteristics. Consequently, tissue spheroids on the membranes expressed high biological activity in terms of oxygen uptake, making them more suitable as building blocks in the fabrication of tissues and organs. Copyright © 2015 John Wiley & Sons, Ltd.

Biofabrication: reappraising the definition of an evolving field.

Biofabrication is an evolving research field that has recently received significant attention. In particular, the adoption of Biofabrication concepts within the field of Tissue Engineering and Regenerative Medicine has grown tremendously, and has been accompanied by a growing inconsistency in terminology. This article aims at clarifying the position of Biofabrication as a research field with a special focus on its relation to and application for Tissue Engineering and Regenerative Medicine. Within this context, we propose a refined working definition of Biofabrication, including Bioprinting and Bioassembly as complementary strategies within Biofabrication.

Post-deposition bioink self-assembly: a quantitative study.

The outcome of a bioprinting process depends on both the deposition of the discrete bioink units and their ability to self-assemble into the desired structure following deposition. Post-printing structure formation is an autonomous process governed by fundamental biological organizing principles. As the quantitative formulation of such principles is notoriously difficult, bioprinting remains largely a trial and error approach. To address this problem, specifically in extrusion bioprinting, we have recently developed an effective computational method, the cellular particle dynamics (CPDs). We have demonstrated the predictive power of CPD in cases of simple printed constructs prepared with spherical multicellular bioink units. Here we generalize CPD to the important practical case of tubular grafts printed with cylindrical bioink units by taking into account the realistic experimental situation in which the length and the volume of the cylinders decrease post-printing. Based on our results, we provide a set of instructions for the use of CPD simulations to directly predict tubular graft formation without the need to carry out the corresponding complex and expensive control experiments. Using these instructions allows the efficient and timely biofabrication of tubular organ structures. A particularly instructive outcome of our analysis is that building tubular organ structures, such as vascular grafts by bioprinting can be done considerably faster by using cylindrical rather than spherical bionk units.

Predictive modeling of post bioprinting structure formation.

Cellular particle dynamics (CPD) is an effective computational method to describe the shape evolution and biomechanical relaxation processes in systems composed of micro tissues such as multicellular aggregates. Therefore, CPD is a useful tool to predict the outcome of postprinting structure formation in bioprinting. The predictive power of CPD has been demonstrated for multicellular systems composed of identical volume-conserving spherical and cylindrical bioink units. Experiments and computer simulations were related through an independently developed theoretical formalism based on continuum mechanics. Here we generalize the CPD formalism to (i) include non-identical bioink particles often used in specific bioprinting applications, (ii) describe the more realistic experimental situation in which during the post-printing structure formation via the fusion of spherical bioink units the volume of the system decreases, and (iii) directly connect CPD simulations to the corresponding experiments without the need of the intermediate continuum theory inherently based on simplifying assumptions.

Biofabrication and testing of a fully cellular nerve graft.

Rupture of a nerve is a debilitating injury with devastating consequences for the individual's quality of life. The gold standard of repair is the use of an autologous graft to bridge the severed nerve ends. Such repair however involves risks due to secondary surgery at the donor site and may result in morbidity and infection. Thus the clinical approach to repair often involves non-cellular solutions, grafts composed of synthetic or natural materials. Here we report on a novel approach to biofabricate fully biological grafts composed exclusively of cells and cell secreted material. To reproducibly and reliably build such grafts of composite geometry we use bioprinting. We test our grafts in a rat sciatic nerve injury model for both motor and sensory function. In particular we compare the regenerative capacity of the biofabricated grafts with that of autologous grafts and grafts made of hollow collagen tubes by measuring the compound action potential (for motor function) and the change in mean arterial blood pressure as consequence of electrically eliciting the somatic pressor reflex. Our results provide evidence that bioprinting is a promising approach to nerve graft fabrication and as a consequence to nerve regeneration.

How cholesterol regulates endothelial biomechanics.

As endothelial cells form the barrier between blood flow and surrounding tissue, many of their functions depend on mechanical integrity, in particular those of the plasma membrane. As component and organizer of the plasma membrane, cholesterol is a regulator of cellular mechanical properties. Disruption of cholesterol balance leads to impairment of endothelial functions and eventually to disease. The mechanical properties of the membrane are strongly affected by the cytoskeleton. As Phosphatidylinositol-4,5-bisphosphate (PIP2) is a key mediator between the membrane and cytoskeleton, it also affects cellular biomechanical properties. Typically, PIP2 is concentrated in cholesterol-rich microdomains, such as caveolae and lipid rafts, which are particularly abundant in the endothelial plasma membrane. We investigated the connection between cholesterol and PIP2 by extracting membrane tethers from bovine aortic endothelial cells (BAEC) at different cholesterol levels and PIP2 conditions. Our results suggest that in BAEC the role of PIP2, as a mediator of membrane-cytoskeleton adhesion, is regulated by cholesterol. Our findings confirm the specific role of cholesterol in endothelial cells and may have implications for cholesterol-dependent vascular pathologies.

Kinetic Monte Carlo and cellular particle dynamics simulations of multicellular systems.

Computer modeling of multicellular systems has been a valuable tool for interpreting and guiding in vitro experiments relevant to embryonic morphogenesis, tumor growth, angiogenesis and, lately, structure formation following the printing of cell aggregates as bioink particles. Here we formulate two computer simulation methods: (1) a kinetic Monte Carlo (KMC) and (2) a cellular particle dynamics (CPD) method, which are capable of describing and predicting the shape evolution in time of three-dimensional multicellular systems during their biomechanical relaxation. Our work is motivated by the need of developing quantitative methods for optimizing postprinting structure formation in bioprinting-assisted tissue engineering. The KMC and CPD model parameters are determined and calibrated by using an original computational-theoretical-experimental framework applied to the fusion of two spherical cell aggregates. The two methods are used to predict the (1) formation of a toroidal structure through fusion of spherical aggregates and (2) cell sorting within an aggregate formed by two types of cells with different adhesivities.

Tissue engineering by self-assembly and bio-printing of living cells.

Biofabrication of living structures with desired topology and functionality requires the interdisciplinary effort of practitioners of the physical, life and engineering sciences. Such efforts are being undertaken in many laboratories around the world. Numerous approaches are pursued, such as those based on the use of natural or artificial scaffolds, decellularized cadaveric extracellular matrices and, most lately, bioprinting. To be successful in this endeavor, it is crucial to provide in vitro micro-environmental clues for the cells resembling those in the organism. Therefore, scaffolds, populated with differentiated cells or stem cells, of increasing complexity and sophistication are being fabricated. However, no matter how sophisticated scaffolds are, they can cause problems stemming from their degradation, eliciting immunogenic reactions and other a priori unforeseen complications. It is also being realized that ultimately the best approach might be to rely on the self-assembly and self-organizing properties of cells and tissues and the innate regenerative capability of the organism itself, not just simply prepare tissue and organ structures in vitro followed by their implantation. Here we briefly review the different strategies for the fabrication of three-dimensional biological structures, in particular bioprinting. We detail a fully biological, scaffoldless, print-based engineering approach that uses self-assembling multicellular units as bio-ink particles and employs early developmental morphogenetic principles, such as cell sorting and tissue fusion.

Computational modeling of epithelial-mesenchymal transformations.

An epithelial-mesenchymal transformation (EMT) involves alterations in cell-cell and cell-matrix adhesion, the detachment of epithelial cells from their neighbors, the degradation of the basal lamina and acquisition of mesenchymal phenotype. Here we present Monte Carlo simulations for a specific EMT in early heart development: the formation of cardiac cushions. Cell rearrangements are described in accordance with Steinberg's differential adhesion hypothesis, which states that cells possess a type-dependent adhesion apparatus and are sufficiently motile to give rise to the tissue conformation with the largest number of strong bonds. We also implement epithelial and mesenchymal cell proliferation, cell type change and extracellular matrix production by mesenchymal cells. Our results show that an EMT is promoted more efficiently by an increase in cell-substrate adhesion than by a decrease in cell-cell adhesion. In addition to cushion tissue formation, the model also accounts for the phenomena of matrix invasion and mesenchymal condensation. We conclude that in order to maintain epithelial integrity during EMT the number of epithelial cells must increase at a controlled rate. Our model predictions are in qualitative agreement with available experimental data.

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

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.

Scaffold-free vascular tissue engineering using bioprinting.

Current limitations of exogenous scaffolds or extracellular matrix based materials have underlined the need for alternative tissue-engineering solutions. Scaffolds may elicit adverse host responses and interfere with direct cell-cell interaction, as well as assembly and alignment of cell-produced ECM. Thus, fabrication techniques for production of scaffold-free engineered tissue constructs have recently emerged. Here we report on a fully biological self-assembly approach, which we implement through a rapid prototyping bioprinting method for scaffold-free small diameter vascular reconstruction. Various vascular cell types, including smooth muscle cells and fibroblasts, were aggregated into discrete units, either multicellular spheroids or cylinders of controllable diameter (300-500 microm). These were printed layer-by-layer concomitantly with agarose rods, used here as a molding template. The post-printing fusion of the discrete units resulted in single- and double-layered small diameter vascular tubes (OD ranging from 0.9 to 2.5mm). A unique aspect of the method is the ability to engineer vessels of distinct shapes and hierarchical trees that combine tubes of distinct diameters. The technique is quick and easily scalable.

Organ printing: tissue spheroids as building blocks.

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.

Relating cell and tissue mechanics: implications and applications.

The Differential Adhesion Hypothesis (DAH) posits that differences in adhesion provide the driving force for morphogenetic processes. A manifestation of differential adhesion is tissue liquidity and a measure for it is tissue surface tension. In terms of this property, DAH correctly predicts global developmental tissue patterns. However, it provides little information on how these patterns arise from the movement and shape changes of cells. We provide strong qualitative and quantitative support for tissue liquidity both in true developmental context and in vitro assays. We follow the movement and characteristic shape changes of individual cells in the course of specific tissue rearrangements leading to liquid-like configurations. Finally, we relate the measurable tissue-liquid properties to molecular entities, whose direct determination under realistic three-dimensional culture conditions is not possible. Our findings confirm the usefulness of tissue liquidity and provide the scientific underpinning for a novel tissue engineering technology.