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.

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.

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.

Adapting bottom-up approaches to tissue engineering is a real challenge. Since the first application of fused deposition modeling for tissue engineering scaffolds, considerable effort has been focused on printing synthetic biodegradable scaffolds. Concurrently a variety of rapid prototyping techniques have been developed to define macroscopically the shapes of deposited biomaterials, including photolithography, syringe-based gel deposition, and solid freeform fabrication. These designed scaffolds have shown promise in regenerating tissues at least equivalent to other scaffolding methods.An exciting advance in scaffold aided tissue regeneration is presented here, that of cell and organ printing, which allows direct printing of c ells and proteins within 3D bydrogel structures. Cell printing opens the possibility to programmed deposition of scaffold structure and cell type, thus controlling the type of tissue that can be regenerated within the scaffold. Several examples of printed tissues will be presented including contractile cardiac hybrids. The hybrid materials have properties that can be tailored in 3D to achieve desired porosities, mechanical and chemical properties. The materials include alginate hydrogels with controlled microshell structures that can be built by spraying cross-linkers onto ungelled alginic acid.Endothelial cells were seen to attach to the inside of these microshells. The cells remained viable in constructs as thick as I cm due to the programmed porosity. Finite element modeling was used to predict the mechanical properties and to generate CAD models with properties matching cardiac tissue. These results suggest that the printing method could be used for hierarchical design of functional cardiac patches, balanced with porosity for mass transport and structural support. (c) 2006 Elsevier B.V. All rights reserved.

Recent advances in organ printing technology for applications relating to medical interventions and organ replacement are described. Organ printing refers to the placement of various cell types into a soft scaffold fabricated according to a computer-aided design template using a single device. Computer aided scaffold topology design has recently gained attention as a viable option to achieve function and mass transport requirements within tissue engineering scaffolds. An exciting advance pioneered in our laboratory is that of simultaneous printing of cells and biomaterials, which allows precise placement of cells and proteins within 3-D hydrogel structures. This advance raises the possibility of spatially controlling not only the scaffold structure, but also the type of tissue that can be grown within the scaffold and the thickness of the tissue as capillaries and vessels could be constructed within the scaffolds. Here we summarize recent advances in printing cells and materials using the same device.

Bioprinting is an evolving tissue engineering technology. It utilizes computer controlled three-dimensional printers for rapid and high-precision construction of three-dimensional biological structures. We employed discrete and continuous bioprinting to build three-dimensional tissue constructs. In the former case bioink particles - spherical cell aggregates composed of many thousands of cells - are delivered one by one into biocompatible scaffolds, the biopaper. Structure formation takes place by the subsequent fusion of the bioink particles due to their liquid-like and self-assembly properties. In the latter case a mixture of cells and scaffold material is extruded from the biocartridge akin to toothpaste to arrive at the desired construct. Specifically, we built rectangular tissue blocks of several hundred microns in thickness as well as tubular structures of several millimeters in height. The physical basis of structure formation was studied by computer simulations.

The process that cardiac cushions undergo to form the mature septa and valves of the adult heart is poorly understood. Periostin is an extracellular molecule that is expressed during cushion mesenchyme formation and throughout valvulogenesis. Once thought to be an osteoblast-specific factor, studies have shown this molecule is antiosteogenic. We have produced an antibody to chicken periostin and examined periostin's localization in the developing avian heart. This antibody recognized proteins from chick heart lysates around 90 kD molecular weight as predicted from the chick periostin mRNA and other periostin orthologs. Periostin immunolocalization was first evident as fibrous strands in the cushion mesenchyme. At HH25, periostin was detected on the basal surface of the trabecular endothelium and also on the endocardial epithelium of the atrioventricular cushion. We hypothesize that periostin may function in the organization of extracellular matrix molecules, providing cues necessary for attachment and spreading during the epithelial-to-mesenchymal transitions of the endocardial epithelium. Enhanced secretion of periostin in the region of delamination may directly or indirectly promote change in the myocardium that precedes or mediates delamination of the leaflet. At later stages of development (HH34-38), periostin was seen predominantly in the fibrous regions of the heart, such as the left atrioventricular valve (LAV), annulus, cardiac skeleton, and adventitia. We propose that periostin is induced by sheer stress and may be an essential molecular component for structures of the heart that undergo mechanical stress or tension during the cardiac cycle.