Developing tissues have intricate, three-dimensional (3D) organizations of cells and extracellular matrix (ECM) that provide the framework necessary to meet morphogenic and necessary demands. Migrating cells, in vivo, are exposed to numerous conflicting signals: chemokines, ECM, growth factors, and physical forces. While most of these have been studied individually in vivo or in vitro, our understanding of how cells integrate these various signals is lacking. We previously developed a novel self-organizing cellularized collagen hydrogel model that is adaptable, tunable, reproducible, and capable of mimicking the multitude of stimuli that cells experience. Our model produced self-assembled toroids of cells that were formed by 24 h. Data we present here show toroids initially form as early as 3 h after seeding. Additionally, toroids formed when cells were seeded on various collagen subtypes and were sensitive to the composition of the hydrogel. Moreover, we found differences in remodeling in toroid gels compared to gels with cells embedded in them using both a collagen binding peptide and rheology. Using scanning electron microscopy, we observed toroids forming a crater-like structure compared to whole gel contractions in mixed in gels. Finally, when multiple cells were mixed prior to seeding, heterogeneous toroids formed with some containing clusters of cells.

During heart valve development, epithelial–mesenchymal transformation (EMT) is a key process for valve formation. EMT leads to the generation of mesenchymal cells that will eventually become the interstitial cells (fibroblasts) of the mature valve. During EMT, cell architecture and motility change markedly; significant changes are also observed in various signaling pathways. Here we systematically examined the expression, localization, and function of zyxin, a focal adhesion protein, in EMT during atrioventricular (AV) valve morphogenesis. Expression and localization studies showed that zyxin was expressed in the AV canal region during crucial stages of valve development. An in vitro 3D collagen gel culture system was used to determine zyxin function either after siRNA gene knockdown or after overexpression. Our studies revealed that zyxin overexpression inhibited endocardial cell migration and cell differentiation and also led to a decrease in the number of migrating mesenchymal cells. Moreover, correlative cytoskeletal changes were apparent in response to both overexpression and knockdown treatments. Thus, zyxin appears to play a role as a regulator of cell migration and differentiation during EMT in chicken AV valve formation.

A need exists to prolong the release of rapidly metabolized peptides of a low molecular weight, while delivering this peptide without environmental interference. Previous studies have used bovine serum albumin (BSA) as a model peptide to study release characteristics from alginate microcapsules. BSA is 66 kDa in size, while the peptide of interest here, connexin-43 carboxyl-terminus mimetic peptide (αCT1), is only 3.4 kDa. Such a change in size results in a much different set of release parameters. Our overall goal is a sustained release over a 24+ h period. Prolonged application of the peptide to a wound site to investigate therapeutic effects is ideal. As a result, a diffusion method using alginate microcapsules, along with the addition of poly-l-lysine and poly-l-ornithine, has been explored. We first aimed to establish and characterize our parameters through a set of parametric tests. Variations in polymer coating, change in pH, and changes in loading ratio have previously been shown to effect release using model compounds. Here we test specific changes in these parameters to show effects on the release of αCT1. Additionally, the microcapsules were attached to several biomaterials and surgical implants by ultraviolet cross-linking to study the effectiveness of attachment and delivery. Analysis and measurements using phase contrast microscopy, scanning electron microscopy, and atomic force microscopy were used to characterize changes in microcapsule morphology.

A novel self-organizing behavior of cellularized gels composed of collagen type 1 that may have utility for tissue engineering is described. Depending on the starting geometry of the tissue culture well, toroidal rings of cells or hollow spheroids were prompted to form autonomously when cells were seeded onto the top of gels and the gels released from attachment to the culture well 12 to 24 h after seeding. Cells within toroids assumed distinct patterns of alignment not seen in control gels in which cells had been mixed in. In control gels, cells formed complex three-dimensional arrangements and assumed relatively higher levels of heterogeneity in expression of the fibronectin splice variant ED-A—a marker of epithelial mesenchymal transformation. The tissue-like constructs resulting from this novel self-organizing behavior may have uses in wound healing and regenerative medicine, as well as building blocks for the iterative assembly of synthetic biological structures.

Extracellular matrix (ECM) plays essential signaling and structural roles required for the proper function of cardiac valves. Cardiac valves initially form as jelly-like cushions, which must adapt to withstand the increased circulation hemodynamics associated with fetal development and birth. This increased biomechanical stability of the developing valves is largely imparted by ECM proteins, which form a highly organized fibrous meshwork. Since heart valve defects contribute to most congenital heart diseases, understanding valve development will provide insight into the pathogenesis of various congenital valve anomalies. Thus, the goal of this study is to describe the spatiotemporal deposition of fibrous ECM proteins during cardiac valve development. Chick embryonic and fetal atrioventricular and semilunar valves were examined by light, confocal, and transmission electron microscopy (TEM). Our data demonstrate that fibrous ECM proteins are deposited when the leaflets are adopting an elongated and compacted phenotype. A general pattern of increased fibrotic ECM deposition was detected in valve tissues. Also, each ECM protein examined displayed a unique pattern of organization, suggesting that regulation of fibrous protein deposition is complex and likely involves both genetic and mechanical factors. In addition, the TEM study revealed the presence of membrane protrusions from valvular endocardium, indicating a potential mechanism for mechanical force transduction.

Development of the epicardium is critical to proper heart formation. It provides all of the precursor cells that form the coronary system and supplies signals that stimulate cardiac myocyte proliferation. The epicardium forms from mesothelial cells associated with the septum transversum and is referred to as the proepicardium (PE). Two different methods by which these PE cells colonize the developing heart have been described. In avians, PE cells form a bridge to the heart over which PE cells migrate onto the heart. In fish and mammals, PE cells form vesicles of cells that detach from the mesothelium, float through the pericardial cavity, and attach to the heart. A previous study of rat PE development investigated this process at the histological level. Protein markers have been developed since this study. Thus, we investigated this important developmental process coupled with these new markers using other visualization techniques such as scanning electron microscopy (SEM) and confocal microscopy. Finally, a novel, three-dimensional (3-D) culture system was used to confirm the identity of the PE cells. In this study, we found convincing evidence that the rat PE cells directly attach to the heart in a manner similar to that observed in avians.

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.