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 to 500 μm). 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 2.5 mm). 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 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.

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Periostin is predominantly expressed in collagen-rich fibrous connective tissues that are subjected to constant mechanical stresses including: heart valves, tendons, perichondrium, cornea, and the periodontal ligament (PDL). Based on these data we hypothesize that periostin can regulate collagen I fibrillogenesis and thereby affect the biomechanical properties of connective tissues. Immunoprecipitation and immunogold transmission electron microscopy experiments demonstrate that periostin is capable of directly interacting with collagen I. To analyze the potential role of periostin in collagen I fibrillogenesis, gene targeted mice were generated. Transmission electron microscopy and morphometric analyses demonstrated reduced collagen fibril diameters in skin dermis of periostin knockout mice, an indication of aberrant collagen I fibrillogenesis. In addition, differential scanning calorimetry (DSC) demonstrated a lower collagen denaturing temperature in periostin knockout mice, reflecting a reduced level of collagen cross-linking. Functional biomechanical properties of periostin null skin specimens and atrioventricular (AV) valve explant experiments provided direct evidence of the role that periostin plays in regulating the viscoelastic properties of connective tissues. Collectively, these data demonstrate for the first time that periostin can regulate collagen I fibrillogenesis and thereby serves as an important mediator of the biomechanical properties of fibrous connective tissues.

A number of properties of certain living embryonic tissues can be explained by considering them as liquids. Tissue fragments left in a shaker bath round up to form spherical aggregates, as do liquid drops. When cells comprising two distinct embryonic tissues are mixed, typically a nucleation-like process takes place, and one tissue sorts out from the other. The equilibrium configurations at the end of such sorting out phenomena have been interpreted in terms of tissue surface tensions arising from the adhesive interactions between individual cells. In the present study we go beyond these equilibrium properties and study the viscoelastic behavior of a number of living embryonic tissues. Using a specifically designed apparatus, spherical cell aggregates are mechanically compressed and their viscoelastic response is followed. A generalized Kelvin model of viscoelasticity accurately describes the measured relaxation curves for each of the four tissues studied. Quantitative results are obtained for the characteristic relaxation times and elastic and viscous parameters. Our analysis demonstrates that the cell aggregates studied here, when subjected to mechanical deformations, relax as elastic materials on short time scales and as viscous liquids on long time scales.

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.

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.

Biofabrication of living structures with desired topology and functionality requires the interdisciplinary effort of practitioners of the physical, life, medical and engineering sciences. Such efforts are being undertaken in many laboratories around the world. Numerous approaches are being pursued, such as those based on the use of natural or artificial scaffolds, decellularized cadaveric extracellular matrices and 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, scaffolds, no matter how sophisticated they are, 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 is 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 bioink particles and employs early developmental morphogenetic principles, such as cell sorting and tissue fusion.