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Three-dimensional (3D) bioprinting, a promising advancement in tissue engineering technology, involves the robotic, layer-by-layer additive biofabrication of functional 3D tissue and organ constructs. This process utilizes biomaterials, typically hydrogels and living cells, following digital models. Traditional tissue engineering uses a classic triad of living cells, scaffolds, and physicochemical signals in bioreactors. A scaffold is a temporary, often biodegradable, support structure. Tissue engineering primarily falls into two categories: (i) scaffold based and (ii) scaffold free. The latter, scaffold-free 3D bioprinting, is gaining increasing popularity. Organ building blocks (OBB), capable of self-assembly and self-organization, such as tissue spheroids, organoids, and assembloids, have begun to be utilized in scaffold-free bioprinting. This article discusses the expanding range of OBB, presents the rapidly evolving collection of bioprinting and bioassembly methods using these OBB, and finally, outlines the advantages, challenges, and future perspectives of using OBB in organ printing.

Biofabrication in space is one of the novel promising and prospective research directions in the rapidly emerging field of space STEM. There are several advantages of biofabrication in space. Under microgravity, it is possible to engineer constructs using more fluidic channels and thus more biocompatible bioinks. Microgravity enables biofabrication of tissue and organ constructs of more complex geometries, thus facilitating novel scaffold-, label-, and nozzle-free technologies based on multi-levitation principles. However, when exposed to microgravity and cosmic radiation, biofabricated tissues could be used to study pathophysiological phenomena that will be useful on Earth and for deep space manned missions. Here, we provide leading concepts about the potential mutual benefits of the application of biofabrication technologies in space., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Bioprinting technologies have been advancing at the convergence of automation, digitalization, and new tissue engineering (TE) approaches. In situ bioprinting may be favored during certain situations when compared with the conventional in vitro bioprinting when de novo tissues are to be printed directly on the intended anatomical location in the living body. To date, few attempts have been made to fabricate in situ tissues, which can be safely arrested and immobilized while printing in preclinical living models. In this review, we have explained the need and utility for in situ bioprinting with regard to the conventional bioprinting approach. The two main in situ bioprinting approaches, namely, robotic arm and handheld approaches, have been defined and differentiated. The various studies involving in situ fabrication of skin, bone, and cartilage tissues have been elucidated. Finally, we have also discussed the advantages, challenges, and the prospects in the field of in situ bioprinting modalities in line with parallel technological advancements. STATEMENT OF SIGNIFICANCE: In situ bioprinting may be favored during certain situations when compared with the conventional in vitro bioprinting when tissues are to be fabricated or repaired directly on the intended anatomical location in the living body, using the body as a bioreactor. However, the technology requires a lot more improvement to fabricate complex tissues in situ, which could eventually be possible through the multi-disciplinary innovations in tissue engineering. This review explains the need and utility and current approaches by handheld and robotic modes for in situ bioprinting. The latest studies involving in situ fabrication of skin, bone, and cartilage tissues have been elucidated. The review also covers the background studies, advantages, technical and ethical challenges, and possible suggestions for future improvements., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Skeletal muscle is a tissue with a complex and hierarchical architecture that influences its functional properties. In order to exert its contractile function, muscle tissue is connected to neural, vascular and connective compartments, comprising finely structured interfaces which are orchestrated by multiple signalling pathways. Pathological conditions such as dystrophies and trauma, or physiological situations such as exercise and aging, modify the architectural organization of these structures, hence affecting muscle functionality. To overcome current limitations of in vivo and standard in vitro models, microfluidics and biofabrication techniques have been applied to better reproduce the microarchitecture and physicochemical environment of human skeletal muscle tissue. In the present review, we aim to critically discuss the role of those techniques, taken individually or in combination, in the generation of models that mimic the complex interfaces between muscle tissue and neural/vascular/tendon compartments. The exploitation of either microfluidics or biofabrication to model different muscle interfaces has led to the development of constructs with an improved spatial organization, thus presenting a better functionality as compared to standard models. However, the achievement of models replicating muscle-tissue interfaces with adequate architecture, presence of fundamental proteins and recapitulation of signalling pathways is still far from being achieved. Increased integration between microfluidics and biofabrication, providing the possibility to pattern cells in predetermined structures with higher resolution, will help to reproduce the hierarchical and heterogeneous structure of skeletal muscle interfaces. Such strategies will further improve the functionality of these techniques, providing a key contribution towards the study of skeletal muscle functions in physiology and pathology.

A long-standing problem in tissue engineering is the biofabrication of perfusable tissue constructs that can be readily connected to the patient's vasculature. It was partially solved by three-dimensional (3D) printing of sacrificial material (e.g., hydrogel) strands: upon incorporation in another cell-laden hydrogel, the strands were removed, leaving behind perfusable channels. Their complexity, however, did not match that of the native vasculature. Here, we propose to use multicellular spheroids as a sacrificial material and investigate their potential benefits in the context of 3D bioprinting of cell aggregates and/or cell-laden hydrogels. Our study is based on computer simulations of postprinting cellular rearrangements. The computational model of the biological system is built on a cubic lattice, whereas its evolution is simulated using the Metropolis Monte Carlo algorithm. The simulations describe structural changes in three types of tissue constructs: a tube made of a single cell type, a tube made of two cell types, and a cell-laden hydrogel slab that incorporates a branching tube. In all three constructs, the lumen is obtained after the elimination of the sacrificial cell population. Our study suggests that sacrificial cell spheroids (sacrospheres) enable one to print tissue constructs outfitted with a finer and more complex network of channels than the ones obtained so far. Moreover, cellular interactions might give rise to a tissue microarchitecture that lies beyond the bioprinter's resolution. Although more expensive than inert materials, sacrificial cells have the potential to bring further progress towards the biofabrication of fully vascularized tissue substitutes.

Tissue engineering (TE) is a highly interdisciplinary research field driven by the goal to restore, replace, or regenerate defective tissues. Throughout more than two decades of intense research, different technological approaches, which can be principally categorized into scaffold-based and scaffold-free strategies, have been developed. In this opinion article, we discuss the emergence of a third strategy in TE. This synergetic strategy integrates the advantages of both of these traditional approaches, while being clearly distinct from them. Its characteristic attributes, numerous practical benefits, and recent literature reports supporting our opinion, are discussed in detail., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The modeling, fabrication, cell loading, and mechanical and in vitro biological testing of biomimetic, interlockable, laser-made, concentric 3D scaffolds are presented. The scaffolds are made by multiphoton polymerization of an organic-inorganic zirconium silicate. Their mechanical properties are theoretically modeled using finite elements analysis and experimentally measured using a Microsquisher(®). They are subsequently loaded with preosteoblastic cells, which remain live after 24 and 72 h. The interlockable scaffolds have maintained their ability to fuse with tissue spheroids. This work represents a novel technological platform, enabling the rapid, laser-based, in situ 3D tissue biofabrication.

Cells respond to topographical, mechanical and biochemical characteristics of the surrounding environment. Capability to reconstruct these factors individually, and also acting in accord, would facilitate systematic investigations of a multitude of related biological and tissue engineering questions. The subject of the present review is a group of technologies allowing realization of customized cell-culture matrices. These methods utilize photochemistry induced by multiphoton absorption and are carried out using essentially identical equipment. Fabrication of 2D microstructured substrates, complex 3D scaffolds, containing actively induced topographies, and immobilization of biomolecules in a spatially defined manner was demonstrated with these techniques. The reviewed reports indicate that multiphoton processing is a promising technology platform for the development of standard biomimetic microenvironments for 3D cell culture.

A mathematical model based on the phase field formulation is developed to study fusion of cellular aggregates/clusters. In a novel biofabrication process known as bioprinting (Mironov et al., 2009a), live multicellular aggregates/clusters are used to make tissue or organ constructs via the layer-by-layer deposition technique, in which the printed bio-constructs are embedded in hydrogels rich in maturogens and placed in bioreactors to undergo the fusion process of self-assembly, maturation, and differentiation to form the desired functional tissue or organ products. We formulate the mathematical model to study the morphological development of the printed bio-constructs during fusion by exploring the chemical-mechanical interaction among the cellular aggregates involved. Specifically, we treat the cellular aggregates and the surrounding hydrogels as two immiscible complex fluids in the time scale comparable to cellular aggregate fusion and then develop an effective mean-field potential that incorporates the long-range, attractive interaction between cells as well as the short-range, repulsive interaction due to immiscibility between the cell and the hydrogel. We then implement the model using a high order spectral method to simulate the making of a set of tissues/organs in simple yet fundamental geometries like a ring, a sheet of tissues, and a Y-shaped, bifurcating vascular junction by the layer-by-layer deposition of spheroidal cellular clusters in the bioprinting technology., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Organ printing, or the layer by layer additive robotic biofabrication of functional three-dimensional tissue and organ constructs using self-assembling tissue spheroid building blocks, is a rapidly emerging technology that promises to transform tissue engineering into a commercially successful biomedical industry. It is increasingly obvious that similar well-established industries implement automated robotic systems on the path to commercial translation and economic success. The use of robotic bioprinters alone however is not sufficient for the development of large industrial scale organ biofabrication. The design and development of a fully integrated organ biofabrication line is imperative for the commercial translation of organ printing technology. This paper presents recent progress and challenges in the development of the essential components of an organ biofabrication line., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Development of methods for scalable biofabrication of uniformly sized tissue spheroids is essential for tissue spheroid-based bioprinting of large size tissue and organ constructs. The most recent scalable technique for tissue spheroid fabrication employs a micromolded recessed template prepared in a non-adhesive hydrogel, wherein the cells loaded into the template self-assemble into tissue spheroids due to gravitational force. In this study, we present an improved version of this technique. A new mold was designed to enable generation of 61 microrecessions in each well of a 96-well plate. The microrecessions were seeded with cells using an EpMotion 5070 automated pipetting machine. After 48 h of incubation, tissue spheroids formed at the bottom of each microrecession. To assess the quality of constructs generated using this technology, 600 tissue spheroids made by this method were compared with 600 spheroids generated by the conventional hanging drop method. These analyses showed that tissue spheroids fabricated by the micromolded method are more uniform in diameter. Thus, use of micromolded recessions in a non-adhesive hydrogel, combined with automated cell seeding, is a reliable method for scalable robotic fabrication of uniform-sized tissue spheroids.

Organ printing or computer-aided robotic layer-by-layer additive biofabrication of thick three-dimensional (3D) living tissue constructs employing self-assembling tissue spheroids is a rapidly evolving alternative to classic solid scaffold-based approaches in tissue engineering. However, the absence of effective methods of accelerated tissue maturation immediately after bioprinting is the main technological imperative and potential impediment for further progress in the development of this emerging organ printing technology. Identification of the optimal combination of factors and conditions that accelerate tissue maturation ('maturogenic' factors) is an essential and necessary endeavour. Screening of maturogenic factors would be most efficiently accomplished using high-throughput quantitative in vitro tissue maturation assays. We have recently reviewed the formation of solid scaffold-free tissue constructs through the fusion of bioprinted tissue spheroids that have measurable material properties. We hypothesize that the fusion kinetics of these tissue spheroids will provide an efficacious in vitro assay of the level of tissue maturation. We report here the results of experimental testing of two simple quantitative tissue spheroid fusion-based in vitro high-throughput screening assays of tissue maturation: (a) a tissue spheroid envelopment assay; and (b) a tissue spheroid fusion kinetics assay., (Copyright © 2010 John Wiley & Sons, Ltd.)

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 possible to engineer 3D thick tissue or organ constructs without effective vascularization. An effective intra-organ vascular system cannot be built by the simple connection of large-diameter vessels and microvessels. Successful engineering of functional human organs suitable for surgical implantation will require concomitant engineering of a 'built in' intra-organ branched vascular system. Organ printing enables biofabrication of human organ constructs with a 'built in' intra-organ branched vascular tree.

Biofabrication can be defined as the production of complex living and non-living biological products from raw materials such as living cells, molecules, extracellular matrices, and biomaterials. Cell and developmental biology, biomaterials science, and mechanical engineering are the main disciplines contributing to the emergence of biofabrication technology. The industrial potential of biofabrication technology is far beyond the traditional medically oriented tissue engineering and organ printing and, in the short term, it is essential for developing potentially highly predictive human cell- and tissue-based technologies for drug discovery, drug toxicity, environmental toxicology assays, and complex in vitro models of human development and diseases. In the long term, biofabrication can also contribute to the development of novel biotechnologies for sustainable energy production in the future biofuel industry and dramatically transform traditional animal-based agriculture by inventing 'animal-free' food, leather, and fur products. Thus, the broad spectrum of potential applications and rapidly growing arsenal of biofabrication methods strongly suggests that biofabrication can become a dominant technological platform and new paradigm for 21st century manufacturing. The main objectives of this review are defining biofabrication, outlining the most essential disciplines critical for emergence of this field, analysis of the evolving arsenal of biofabrication technologies and their potential practical applications, as well as a discussion of the common challenges being faced by biofabrication technologies, and the necessary conditions for the development of a global biofabrication research community and commercially successful biofabrication industry.

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.

Centrifugal casting allows rapid biofabrication of tubular tissue constructs by suspending living cells in an in situ cross-linkable hydrogel. We hypothesize that introduction of laser-machined micropores into a decellularized natural scaffold will facilitate cell seeding by centrifugal casting and increase hydrogel retention, without compromising the biomechanical properties of the scaffold. Micropores with diameters of 50, 100, and 200 mum were machined at different linear densities in decellularized small intestine submucosa (SIS) planar sheets and tubular SIS scaffolds using an argon laser. The ultimate stress and ultimate strain values for SIS sheets with laser-machined micropores with diameter 50 mum and distance between holes as low as 714 mum were not significantly different from unmachined control SIS specimens. Centrifugal casting of GFP-labeled cells suspended in an in situ cross-linkable hyaluronan-based hydrogel resulted in scaffold recellularization with a high density of viable cells inside the laser-machined micropores. Perfusion tests demonstrated the retention of the cells encapsulated within the HA hydrogel in the microholes. Thus, an SIS scaffold with appropriately sized microholes can be loaded with hydrogel encapsulated cells by centrifugal casting to give a mechanically robust construct that retains the cell-seeded hydrogel, permitting rapid biofabrication of tubular tissue construct in a "bioreactor-free" fashion.

The existing methods of biofabrication for vascular tissue engineering are still bioreactor-based, extremely expensive, laborious and time consuming and, furthermore, not automated, which would be essential for an economically successful large-scale commercialization. The advances in nanotechnology can bring additional functionality to vascular scaffolds, optimize internal vascular graft surface and even help to direct the differentiation of stem cells into the vascular cell phenotype. The development of rapid nanotechnology-based methods of vascular tissue biofabrication represents one of most important recent technological breakthroughs in vascular tissue engineering because it dramatically accelerates vascular tissue assembly and, importantly, also eliminates the need for a bioreactor-based scaffold cellularization process.

Understanding the principles of biological self-assembly is indispensable for developing efficient strategies to build living tissues and organs. We exploit the self-organizing capacity of cells and tissues to construct functional living structures of prescribed shape. In our technology, multicellular spheroids (bio-ink particles) are placed into biocompatible environment (bio-paper) by the use of a three-dimensional delivery device (bio-printer). Our approach mimics early morphogenesis and is based on the realization that the genetic control of developmental patterning through self-assembly involves physical mechanisms. Three-dimensional tissue structures are formed through the postprinting fusion of the bio-ink particles, in analogy with early structure-forming processes in the embryo that utilize the apparent liquid-like behavior of tissues composed of motile and adhesive cells. We modeled the process of self-assembly by fusion of bio-ink particles, and employed this novel technology to print extended cellular structures of various shapes. Functionality was tested on cardiac constructs built from embryonic cardiac and endothelial cells. The postprinting self-assembly of bio-ink particles resulted in synchronously beating solid tissue blocks, showing signs of early vascularization, with the endothelial cells organized into vessel-like conduits.

Casting is a process by which a material is introduced into a mold while it is liquid, allowed to solidify in a predefined shape inside the mold, and then removed to give a fabricated object, part or casing. Centrifugal casting could be defined as a process of molding using centrifugal forces. Although the centrifugal casting technology has a long history in metal manufacturing and in the plastics industry, only recently has this technology attracted the attention of tissue engineers. Initially, centrifugation was used to optimize cell seeding on a solid scaffold. More recently, centrifugal casting has been used to create tubular scaffolds and both tubular and flat multilayered, living tissue constructs. These newer applications were enabled by a new class of biocompatible in situ crosslinkable hydrogels that mimic the extracellular matrix. Herein the authors summarize the state of the art of centrifugal casting technology in tissue engineering, they outline associated technological challenges, and they discuss the potential future for clinical applications.

Organ printing or biomedical application of rapid prototyping, also defined as additive layer-by-layer biomanufacturing, is an emerging transforming technology that has potential for surpassing traditional solid scaffold-based tissue engineering. Organ printing has certain advantages: it is an automated approach that offers a pathway for scalable reproducible mass production of tissue engineered products; it allows a precised simultaneous 3D positioning of several cell types; it enables creation tissue with a high level of cell density; it can solve the problem of vascularization in thick tissue constructs; finally, organ printing can be done in situ. The ultimate goal of organ-printing technology is to fabricate 3D vascularized functional living human organs suitable for clinical implantation. The main practical outcomes of organ-printing technology are industrial scalable robotic biofabrication of complex human tissues and organs, automated tissue-based in vitro assays for clinical diagnostics, drug discovery and drug toxicity, and complex in vitro models of human diseases. This article describes conceptual framework and recent developments in organ-printing technology, outlines main technological barriers and challenges, and presents potential future practical applications.

The First Annual Charleston Bioprinting Symposium was organized by the Bioprinting Research Center of the Medical University of South Carolina (MUSC) and convened July 21, 2006, in Charleston, South Carolina. In broad terms, bioprinting is the application of rapid prototyping technology to the biomedical field. More specifically, it is defined as the layer by layer deposition of biologically relevant material. The 2006 Symposium included four sessions: Computer-aided design and Bioprinting, Bioprinting Technologies; Hydrogel for Bioprinting and, finally, a special session devoted to ongoing research projects at the MUSC Bioprinting Research Center. The Symposium highlight was the presentation of the multidisciplinary Charleston Bioengineered Kidney Project. This symposium demonstrated that bioprinting or robotic biofabrication is one of the most exciting and fast-emerging branches in the tissue engineering field. Robotic biofabrication will eventually lead to industrial production of living human organs suitable for clinical transplantation. The symposium demonstrated that although there are still many technological challenges, organ printing is a rapidly evolving feasible technology.

The goal of Charleston Bioengineered Kidney Project is to engineer a functional living human kidney suitable for surgical implantation using principles of directed tissue self-assembly and tissue fusion. This is a multidisciplinary project which incorporates multiple innovative bioengineering technologies and expertise from a broad spectrum of disciplines. The conceptual framework, engineering principles, design, potential cell source as well as the first preliminary data demonstrating the feasibility of the proposed Charleston Bioengineered Kidney Project are outlined. The potential challenges are described. Finally, the experts' opinion about the proposed project is also presented.

Proepicardial/epicardial-derived cells are the main origin of the early embryonic coronary vascular bed. In vivo coronary vasculogenesis, which is a fast-occurring event, can be mimicked in vitro by culturing proepicardial tissue in different ways. The in vitro vasculogenic model presented in this study (a proepicardial suspension culture assay) partially reproduces coronary vascular development from its cellular precursors, a process known to be highly dependent on cell migration, cell differentiation, cell adhesion/sorting, and tissue fusion phenomena. The main aim of this study is to study the triggering signals and the cellular dynamics that regulate the differentiation of proepicardial cells into the angioblastic/endothelial lineage and their in vitro vasculogenic potential. Our results indicate that hanging drop-cultured proepicardia, which have an intrinsic vascular potential, behave like self-assembling cell aggregates or spheroids that can fuse to give rise to complex vascularized 3D structures. We believe that these self-assembling cell aggregates are an optimal choice to study the differentiation of coronary angioblasts, as well as a good method to reproduce vascular development in vitro. Finally, we propose the proepicardium as a suitable cellular source for vascular tissue engineering.

Several years ago, the American Association of Anatomists (AAA) launched an innovative mini-meeting format as part of their annual meeting. The AAA continued this tradition by sponsoring a 2-day mini-symposium as a part of its meeting at FASEB Experimental Biology, 2006 in San Francisco, CA, USA. This year, the mini-symposium was focused on the promising and rapidly developing field of 'regenerative medicine'. The mini-symposium on 'regenerative medicine' included four separate but thematically integrated sessions: stem cells for regenerative medicine; biomimetic matrices for regenerative medicine; endothelial-mesenchymal transformation in cardiovascular regenerative medicine; and tissue engineering technologies for regenerative medicine. The goal of these sessions was to identify progress and highlight new trends and directions in the evolving field of regenerative medicine. It was an exciting 2-day mini-symposium that reviewed the differential potential of embryonic and adult stem cells, their role in tissue turnover and possible applications in tissue regeneration; identified important evolving basic science issues, such as the role of endothelial-mesenchymal transformation and stem cell recruitment in cardiovascular regenerative medicine; and, finally, clearly demonstrated how understanding basic scientific principles can be translated into novel cell therapeutics and tissue engineering modalities. The workshop also demonstrated the multidisciplinary (speakers included stem cell and developmental biologists, chemical engineers, tissue engineers, biophysicists, mathematicians and surgeons) and international (speakers represented US, Japan, Canada, Switzerland and Korea) character of ongoing efforts in the area of regenerative medicine and stem cell biology, impressive progress in this field, and confirmed the strong potential for clinical translation of emerging regenerative medicine technologies.

An increasing demand for directed assembly of biologically relevant materials, with prescribed three-dimensional hierarchical organizations, is stimulating technology developments with the ultimate goal of re-creating multicellular tissues and organs de novo. Existing techniques, mostly adapted from other applications or fields of research, are capable of independently meeting partial requirements for engineering biological or biomimetic structures, but their integration toward organ engineering is proving difficult. Inspired by recent developments in material transfer processes operating at all relevant length scales--from nano to macro--which are amenable to biological elements, a new research field of bioprinting and biopatterning has emerged. Here we present a short review regarding the framework, state of the art, and perspectives of this new field, based on the findings presented at a recent international workshop.

Tissue engineering is a fast-evolving field of biomedical science and technology with future promise to manufacture living tissues and organs for replacement, repair, and regeneration of diseased organs. Owing to the specific role of hemodynamics in the development, maintenance, and functioning of the cardiovascular system, bioreactors are a fundamental of cardiovascular tissue engineering. The development of perfusion bioreactor technology for cardiovascular tissue engineering is a direct sequence of previous historic successes in extracorporeal circulation techniques. Bioreactors provide a fluidic environment for tissue engineered tissue and organs, and guarantee their viability, maturation, biomonitoring, testing, storage, and transportation. There are different types of bioreactors and they vary greatly in their size, complexity, and functional capabilities. Although progress in design and functional properties of perfusion bioreactors for tissue engineered blood vessels, heart valves, and myocardial patches is obvious, there are some challenges and insufficiently addressed issues, and room for bioreactor design improvement and performance optimization. These challenges include creating a triple perfusion bioreactor for vascularized tubular tissue engineered cardiac construct; designing and manufacturing fluidics-based perfused minibioreactors; incorporation of systematic mathematical modeling and computer simulation based on computational fluid dynamics into the bioreactor designing process; and development of automatic systems of hydrodynamic regime control. Designing and engineering of built-in noninvasive biomonitoring systems is another important challenge. The optimal and most efficient perfusion and conditioning regime, which accelerates tissue maturation of tissue-engineered constructs also remains to be determined. This is a first article in a series of reviews on critical elements of cardiovascular tissue engineering technology describing the current status, unsolved problems, and challenges of bioreactor technology in cardiovascular tissue engineering and outlining future trends and developments.

Achieving the optimal cell density and desired cell distribution in scaffolds is a major goal of cell seeding technologies in tissue engineering. In order to reach this goal, a novel centrifugal casting technology was developed using in situ crosslinkable hyaluronan-based (HA) synthetic extracellular matrix (sECM). Living cells were suspended in a viscous solution of thiol-modified HA and thiol-modified gelatin, a polyethyleneglycol diacrylate crosslinker was added, and a hydrogel was formed during rotation. The tubular tissue constructs consisting of a densely packed cell layer were fabricated with the rotation device operating at 2000 rpm for 10 min. The majority of cells suspended in the HA mixture before rotation were located inside the layer after centrifugal casting. Cells survived the effect of the centrifugal forces experienced under the rotational regime employed. The volume cell density (65.6%) approached the maximal possible volume density based on theoretical sphere packing models. Thus, centrifugal casting allows the fabrication of tubular constructs with the desired redistribution, composition and thickness of cell layers that makes the maximum efficient use of available cells. Centrifugal casting in this sECM would enable rapid fabrication of tissue-engineered vascular grafts, as well as other tubular and planar tissue-engineered constructs.

The Second International Workshop on Bioprinting, Biopatterning and Bioassembly was held at the Medical University of South Carolina (MUSC), located in the beautiful, historic city of Charleston. The workshop attracted > 50 participants from 10 different countries, including mechanical and chemical engineers, molecular, cell and developmental biologists, biophysicists, mathematicians, clinicians, humanists and artists. Bioprinting can be defined as computer-aided, automatic, layer-by-layer deposition, transfer and patterning of biologically relevant materials. The workshop goal was to gather the world's experts and leaders, present the latest results, assess future trends, explore new applications, and promote international collaborations and academic-industrial partnerships. The workshop demonstrated the multidisciplinary and global character of ongoing efforts in the development of bioprinting technology, galvanised an evolving community of bioprintists, and demonstrated feasibility as well as strong potential for a broad spectrum of applications of bioprinting technology. The Third International Workshop on Bioprinting, Biopatterning and Bioassembly is planned for Japan in 2006.

Aggregates of living cells (i.e. model tissue fragments) under appropriate conditions fuse like liquid drops. According to Steinberg's differential adhesion hypothesis (DAH), this may be understood by assuming that cells are motile and tissues made of such cells possess an effective surface tension. Here we show that based on these properties three-dimensional cellular structures of prescribed shape can be constructed by a novel method: cell aggregate printing. Spherical aggregates of similar size made of cells with known adhesive properties were prepared. Aggregates were embedded into biocompatible gels. When the cellular and gel properties, as well as the symmetry of the initial configuration were appropriately adjusted the contiguous aggregates fused into ring-like organ structures. To elucidate the driving force and optimal conditions for this pattern formation, Monte Carlo simulations based on a DAH motivated model were performed. The simulations reproduced the experimentally observed cellular arrangements and revealed that the control parameter of pattern evolution is the gel-tissue interfacial tension, an experimentally accessible parameter.

Arterial growth during embryonic vascular development is associated with longitudinal strain. The longitudinal strain is an important element of the embryonic vascular mechanical environment (EVME). Thus, a perfusion bioreactor for vascular tissue engineered constructs must include the functional capacity for longitudinal strain. To accomplish this goal, a perfusion bioreactor with the capacity for longitudinal strain was developed. The bioreactor includes two media perfusion systems: one for the inside perfusion and one for the outside perfusion of the cardiovascular engineered tubular construct (CETC). The watertight perfusion chamber allows periodic changing of longitudinal strain of the construct during mechanical conditioning. The range of the longitudinal strain is 0% to 200%. The biomechanical properties of the CETC are controlled by a pressure transducer and a digital TV camera. The pressure transducer and TV camera are connected to a computer. This allows the recording of a relationship between the radius of the VTEG and pressure in both static and dynamic regimens. This bioreactor can perform biomechanical conditioning with longitudinal strain.

Tissue engineering technology promises to solve the organ transplantation crisis. However, assembly of vascularized 3D soft organs remains a big challenge. Organ printing, which we define as computer-aided, jet-based 3D tissue-engineering of living human organs, offers a possible solution. Organ printing involves three sequential steps: pre-processing or development of "blueprints" for organs; processing or actual organ printing; and postprocessing or organ conditioning and accelerated organ maturation. A cell printer that can print gels, single cells and cell aggregates has been developed. Layer-by-layer sequentially placed and solidified thin layers of a thermo-reversible gel could serve as "printing paper". Combination of an engineering approach with the developmental biology concept of embryonic tissue fluidity enables the creation of a new rapid prototyping 3D organ printing technology, which will dramatically accelerate and optimize tissue and organ assembly.

Aggregates of pre-sorted cells form structures called tissue spheroids that have been widely used in the field of tissue engineering. The greater contact of the cells with the culture medium is directly related to cell viability and to the increase of proliferation rate. Due to the characteristics of a 3D environment, at some zones within the tissue spheroids some cells are not equally exposed to the environment, impacting in the formation of microenvironments with decreased oxygen, nutrients and soluble factors produced by cellular metabolism leading to the formation of low proliferation areas and consequently necrosis (cell death). The fusion of cells also changes the catabolites flow, generating a very heterogeneous diffusion. The idea of this project is to develop and improve a microscaffold based on the concept of lockyballs that have cell support function for tissue engineering. Lockyballs have hooks and loops which attach each other assuring a 3D cellularized mesh. The model has a spherical outer structure and inner hollow microsphere constituted by pores with diameters smaller than the cell ones, whose function would be to prevent cell entry and provide an environment suitable to diffusion gradient necessary for the cell viability of the spheroid avoiding necrosis. The first stage of the work consisted in the generation of different three dimensional models by Computer Aided Design (CAD) software Rhinoceros 5.0. At the second stage, the CAD model was imported into finite element method (FEM) software (ANSYS R16 Static) to perform computational simulation, which consisted of structural analyses. Computational simulations were essential to predict the diffusion phenomenon inside the whole 3D structure. The development of new microscaffolds models can enhance the regenerative capacity and 3D tissues construction. [ABSTRACT FROM AUTHOR]

Abstract: Organ printing technology or robotic additive biofabrication of 3D functional tissue and organ constructs is based on using tissue spheroids as building blocks. In order to bioprint human organs it is necessary to develop technology for scalable production of millions tissue spheroids. Ideally, these tissue spheroids must have standard size and shape suitable for bioprinting process. The scalable biofabrication of large volume of standard size tissue spheroids could be achieved only by maximal employment of robotics and automation technologies. The three main competing groups of emerging tissue spheroid biofabrication technologies include: i) modified handing drop method, 2) molded non-adhesive hydrogel technology and iii) digital microfluidic technologies. The comparative analysis of emerging scalable tissue spheroid biofabrication technologies has been performed. Our data indicates that all these technologies have potential for robotization and automation. The molded non-adhesive hydrogel technologies provide best outcome for standardization of tissue spheroid size. The microfluidics technology has strong advantage in accelerating of tissue spheroids biofabrication (theoretically, up to 10 000 droplets per second). New emerging approaches for biofabrication tissue spheroid using nanopatterned biomimetic surface and technologies for tissue spheroid encapsulation and functionalization will be also presented. The tissue spheroid biofabrication technologies are still evolving and represent hot area in biofabrication research. Moreover, these technologies are already subject of ongoing commercialization. Thus, it is safe to predict that scalable robotic tissue spheroid biofabricators must be integrated parts of organ biofabrication line. [Copyright &y& Elsevier]