Collagen-embedded islets drove a small (albeit not significant) shift toward a proangiogenic CD206MHCII(M2-like) macrophage response, which was a feature of module-associated vascularization. While these results open the potential for using s.c. islet delivery as a treatment option for type I diabetes, the more immediate benefit may be for the exploration of revascularized islet biology.
Modular tissue engineering is a method of building vascularized tissue-engineered constructs. Submillimeter-sized collagen pieces (modules) coated with a layer of endothelial cells (EC; vascular component), and with embedded functional cells, are self-assembled into a larger, three-dimensional tissue. In this study, we examined the use of developmental endothelial locus-1 (Del-1), an extracellular matrix protein with proangiogenic properties, as a means of tipping the angiogenic balance in human umbilical vein endothelial cells incorporated in modular tissue-engineered constructs. The motivation was to enhance the vascularization of these constructs upon transplantation in vivo, in this case, without the use of exogenous mesenchymal stromal cells. EC were transduced using a lentiviral construct to overexpress Del-1. The Del-1 EC formed more sprouts in a fibrin gel sprouting assay in vitro compared with eGFP (control) transduced EC, as expected. Del-1 EC had a distinct profile of gene expression (upregulation of matrix metalloproteinase-9 [MMP-9], urokinase-type plasminogen activator [uPA/PLAU], vascular endothelial growth factor [VEGF-A], and intercellular adhesion molecule-1 [ICAM-1]; downregulation of angiopoietin-2 [Ang2]), also supporting the notion of "tipping the angiogenic balance". On the other hand, contrary to our expectations, when Del-1 EC-coated modules were implanted subcutaneously in a severe combined immunodeficient/beige animal model, the proangiogenic effect of Del-1 was less remarkable. There was only a small increase in the number of blood vessels formed in Del-1 implants compared with the eGFP implants, and only few blood vessels formed at the implant site in both cases. This was presumed due to limited EC survival after transplantation. We speculate that if we could improve EC survival in our study (for example, by adding other prosurvival factors or supporting cells), we would see a greater Del-1-induced angiogenic benefit in vivo as a consequence of increased Del-1 secretion by a higher number of surviving cells.
The lack of vascularization limits the creation of engineered tissue constructs with clinically relevant sizes. We pioneered a bottom-up process (modular tissue engineering) in which constructs with intrinsic vasculature were assembled from endothelialized building blocks. In this study, we prepared an interpenetrating polymer network (IPN) hydrogel from a collagenalginate blend and evaluated its use as microspheres in modular tissue engineering. Ionotropic gelation of alginate was combined with collagen fibrillogenesis, and the resulting hydrogel was stiffer and had greater resistance to enzymatic degradation relative to that of collagen alone; the viability of embedded mesenchymal stromal cells (adMSC) was unaltered. IPN microspheres were fabricated by a coaxial air-flow technique, and an additional step of collagen coating was required to have human umbilical vein endothelial cells (HUVEC) attach and proliferate. When implanted subcutaneously in SCID/bg mice, adMSC-HUVEC microspheres promoted more blood vessels at day 7 relative to microspheres without adMSC but coated with HUVEC. Perfusion studies confirmed that these vessels were connected to the host vasculature. Fewer vessels were detected in both groups at day 21, but in adMSC-HUVEC explants, more smooth muscle cells had wrapped around vessels, and CLARITY processing of whole explants revealed a restricted leakage of blood. The capacity for rapid gelation and high throughput production are promising features for the use of these microspheres in modular tissue engineering.
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