Paracrine signals, essential for the proper survival and functioning of tissues, can be mimicked by delivery of therapeutic proteins within engineered tissue constructs. Conventional delivery methods are of limited duration and are unresponsive to the local environment. I developed a system for sustained and regulated delivery of paracrine signals by encapsulating living cells of one type in alginate beads and co-suspending these cell-loaded particles along with unencapsulated cells of a second type within a 3D protein gel. By using cells as particulate protein delivery sources in a 3D gel, an array of soluble factors are delivered in an adaptable manner throughout the gel material for as long as the cells are alive and healthy.
This system was applied to vascular tissue engineering by placing human placental microvascular pericytes (PCs) in the particulate alginate phase and human umbilical vein endothelial cells (HUVECs) in the protein gel phase. Particle characteristics were optimized to keep the encapsulated PCs viable for at least two weeks. Encapsulated PCs were bioactive in vitro, secreting multiple angiogenic proteins for up to 7 days, including hepatocyte growth factor and vascular endothelial growth factor, and responding to externally applied HUVEC-derived signals. Medium conditioned by encapsulated PCs stimulated the formation of longer cumulative sprout lengths (1.4x) in an HUVEC sprouting assay.
Encapsulated PCs were biologically active in the complete dual-cell system described. These encapsulated PCs influenced HUVEC behavior in the surrounding gel by enhancing the formation of vessel-like structures, when compared to empty alginate bead controls. Significantly more multi-cell cords (3.1x) and tubes (2.6x) were formed by HUVEC in the presence of encapsulated PC. Additionally, multi-cell cords were significantly longer (1.4x) and lumen diameter was significantly smaller (1.5x) than in control gels containing empty alginate particles.
Encapsulated PCs appear to lead to important functional consequences in the developing vascular network: paracrine signals from the entrapped PCs lead to smaller lumen diameters in vessels formed by HUVEC that were suspended in protein gels and subcutaneously implanted in the abdominal wall of immunodeficient mice. In native in vivo microvessels, PCs reside in the basement membrane and directly contact EC tubes. Previous studies, in which EC-PC contact was permitted, showed that the presence of PCs lead to smaller vessel diameter. These earlier studies hypothesize that this limitation of lumen diameter requires EC-PC contact, and was due to a physical restriction. Our studies, on the other hand, suggest that paracrine signals contribute to this control of vessel diameter.
I conclude that alginate-encapsulated cells can provide functional paracrine signals within engineered tissues. The dual-cell system described in this dissertation can be used both as an engineering therapy to adaptably deliver an array of paracrine signals and as a platform for studying the purely paracrine interactions between two cell types in the absence of cell-cell contact.
|Advisor:||Saltzman, W. Mark|
|School Location:||United States -- Connecticut|
|Source:||DAI-B 75/09(E), Dissertation Abstracts International|
|Subjects:||Biology, Biomedical engineering, Medicine|
|Keywords:||Alginate, Cell encapsulation, Co-culture, Pericycle, Protein delivery, Vascular tissue engineering|
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