Co-cultures of endothelial cells (EC) and mesenchymal stem cells (MSC) in three-dimensional (3D) protein hydrogels can be used to recapitulate aspects of vasculogenesis in vitro. MSC provide paracrine signals that stimulate EC to form vessel-like structures, which mature as the MSC transition to the role of mural cells. In this study, vessel-like network formation was studied using 3D collagen/fibrin (COL/FIB) matrices seeded with embedded EC and MSC and cultured for 7 days. The EC:MSC ratio was varied from 5:1, 3:2, 1:1, 2:3 and 1:5. The matrix composition was varied at COL/FIB compositions of 100/0 (pure COL), 60/40, 50/50, 40/60 and 0/100 (pure FIB). Vasculogenesis was markedly decreased in the highest EC:MSC ratio, relative to the other cell ratios. Network formation increased with increasing fibrin content in composite materials, although the 40/60 COL/FIB and pure fibrin materials exhibited the same degree of vasculogenesis. EC and MSC were co-localized in vessel-like structures after 7 days and total cell number increased by approximately 70%. Mechanical property measurements showed an inverse correlation between matrix stiffness and network formation. The effect of matrix stiffness was further investigated using gels made with varying total protein content and by crosslinking the matrix using the dialdehyde glyoxal. This systematic series of studies demonstrates that matrix composition regulates vasculogenesis in 3D protein hydrogels, and further suggests that this effect may be caused by matrix mechanical properties. These findings have relevance to the study of neovessel formation and the development of strategies to promote vascularization in transplanted tissues.

As tissue engineering products move toward the clinic, nondestructive methods to monitor their development and ensure quality are needed. In this study, high-resolution spectral ultrasound imaging (SUSI) was used to noninvasively characterize mineral content in collagen hydrogels. SUSI was used to generate three-dimensional (3D) grayscale (GS) images of construct morphology with submillimeter resolution. Spectral analysis of the backscattered radio frequency (RF) ultrasound signals was used to determine the midband fit (MBF) and slope of the linearized RF spectrum. These parameters are operator and instrument independent, and were used to characterize the spatial distribution of mineral in constructs supplemented with hydroxyapatite particles. GS and MBF correlated closely with mineral content, while slope was not dependent on concentration. SUSI also was used to monitor mineralization of collagen constructs by immersion in simulated body fluid (SBF) over 21 days. The construct surface was mineralized before the interior, and there was a dose-dependent effect of SBF concentration on degree of mineralization and deposited particle size. MBF density was closely correlated with the amount of calcium deposited. These data demonstrate that SUSI has utility as a noninvasive imaging method for quantitative analysis of mineralization in 3D protein constructs. Such techniques may assist the development of engineered orthopedic tissues.

The process of new blood vessel formation is critical in tissue development, remodeling and regeneration. Modular tissue engineering approaches have been developed to enable the bottom-up assembly of more complex tissues, including vascular networks. In this study, collagen-fibrin composite microbeads (100-300 μm in diameter) were fabricated using a water-in-oil emulsion technique. Human endothelial cells and human fibroblasts were embedded directly in the microbead matrix at the time of fabrication. Microbead populations were characterized and cultured for 14 days either as free-floating populations or embedded in a surrounding fibrin gel. The collagen-fibrin matrix efficiently entrapped cells and supported their viability and spreading. By 7 days in culture, endothelial cell networks were evident within microbeads, and these structures became more prominent by day 14. Fibroblasts co-localized with endothelial cells, suggesting a pericyte-like function, and laminin deposition indicated maturation of the vessel networks over time. Microbeads embedded in a fibrin gel immediately after fabrication showed the emergence of cells and the coalescence of vessel structures in the surrounding matrix by day 7. By day 14, inosculation of neighboring cords and prominent vessel structures were observed. Microbeads pre-cultured for 7 days prior to embedding in fibrin gave rise to vessel networks that emanated radially from the microbead by day 7, and developed into connected networks by day 14. Lumen formation in endothelial cell networks was confirmed using confocal sectioning. These data show that collagen-fibrin composite microbeads support vascular network formation. Microbeads embedded directly after fabrication emulated the process of vasculogenesis, while the branching and joining of vessels from pre-cultured microbeads resembled angiogenesis. This modular microtissue system has utility in studying the processes involved in new vessel formation, and may be developed into a therapy for the treatment of ischemic conditions.

For nearly 60 years, the ATP activation and the CTP inhibition of Escherichia coli aspartate transcarbamoylase (ATCase) has been the textbook example of allosteric regulation. We present kinetic data and five X-ray structures determined in the absence and presence of a Mg(2+) concentration within the physiological range. In the presence of 2 mM divalent cations (Mg(2+), Ca(2+), Zn(2+)), CTP does not significantly inhibit the enzyme, while the allosteric activation by ATP is enhanced. The data suggest that the actual allosteric inhibitor of ATCase in vivo is the combination of CTP, UTP, and a divalent cation, and the actual allosteric activator is a divalent cation with ATP or ATP and GTP. The structural data reveals that two NTPs can bind to each allosteric site with a divalent cation acting as a bridge between the triphosphates. Thus, the regulation of ATCase is far more complex than previously believed and calls many previous studies into question. The X-ray structures reveal that the catalytic chains undergo essentially no alternations; however, several regions of the regulatory chains undergo significant structural changes. Most significant is that the N-terminal region of the regulatory chains exists in different conformations in the allosterically activated and inhibited forms of the enzyme. Here, a new model of allosteric regulation is proposed.