Nanoparticulate delivery of agents for induced elastogenesis in three-dimensional collagenous matrices.

The degradation of elastic matrix in the infrarenal aortic wall is a critical parameter underlying the formation and progression of abdominal aortic aneurysms. It is mediated by the chronic overexpression of matrix metalloprotease (MMP)-2 and MMP-9, leading to a progressive loss of elasticity and weakening of the aortic wall. Delivery of therapeutic agents to inhibit MMPs, while concurrently coaxing cell-based regenerative repair of the elastic matrix represents a potential strategy for slowing or arresting abdominal aortic aneurysm growth. Previous studies have demonstrated elastogenic induction of healthy and aneurysmal aortic smooth muscle cells and inhibition of MMPs, following exogenous delivery of elastogenic factors such as transforming growth factor (TGF)-ß1, as well as MMP-inhibitors such as doxycycline (DOX) in two-dimensional culture. Based on these findings, and others that demonstrated elastogenic benefits of nanoparticulate delivery of these agents in two-dimensional culture, poly(lactide-co-glycolide) nanoparticles were developed for localized, controlled and sustained delivery of DOX and TGF-ß1 to human aortic smooth muscle cells within a three-dimensional gels of type I collagen, which closely simulate the arterial tissue microenvironment. DOX and TGF-ß1 released from these nanoparticles influenced elastogenic outcomes positively within the collagen constructs over 21 days of culture, which were comparable to that induced by exogenous supplementation of DOX and TGF-ß1 within the culture medium. However, this was accomplished at doses ~20-fold lower than the exogenous dosages of the agents, illustrating that their localized, controlled and sustained delivery from nanoparticles embedded within a three-dimensional scaffold is an efficient strategy for directed elastogenesis. Copyright © 2014 John Wiley & Sons, Ltd.

Impact of cyclic stretch on induced elastogenesis within collagenous conduits.

In vitro tissue engineering of vascular conduits requires a synergy between several external factors, including biochemical supplementation and mechanotranductive stimulation. The goal of this study was to improve adult human vascular smooth muscle cell orientation and elastic matrix synthesis within 3D tubular collagen gel constructs. We used a combination of elastogenic factors (EFs) previously tested in our lab, along with cyclic circumferential strains at low amplitude (2.5%) delivered at a range of frequencies (0.5, 1.5, and 3 Hz). After 21 days of culture, the constructs were analyzed for elastic matrix outcomes, activity of matrix metalloproteinases (MMPs)-2 and -9, cell densities and phenotype, and mechanical properties of constructs. While cell densities remained unaffected by the addition of stretch, contractile phenotypic markers were elevated in all stretched constructs relative to control. Constructs cultured with EFs stretched at 1.5 Hz exhibited the maximum elastin mRNA expression and total matrix elastin (over sixfold vs. the static EFs control). MMP-2 content was comparable in all treatment conditions, but MMP-9 levels were elevated at the higher frequencies (1.5 and 3 Hz). Minimal circumferential orientation was achieved and the mechanical properties remained comparable among the treatment conditions. Overall, constructs treated with EFs and stretched at 1.5 Hz exhibited the most elastogenic outcomes.

Tissue engineering and regenerative strategies to replicate biocomplexity of vascular elastic matrix assembly.

Cardiovascular tissues exhibit architecturally complex extracellular matrices, of which the elastic matrix forms a major component. The elastic matrix critically maintains native structural configurations of vascular tissues, determines their ability to recoil after stretch, and regulates cell signaling pathways involved in morphogenesis, injury response, and inflammation via biomechanical transduction. The ability to tissue engineer vascular replacements that incorporate elastic matrix superstructures unique to cardiac and vascular tissues is thus important to maintaining vascular homeostasis. However, the vascular elastic matrix is particularly difficult to tissue engineer due to the inherently poor ability of adult vascular cells to synthesize elastin precursors and organize them into mature structures in a manner that replicates the biocomplexity of elastic matrix assembly during development. This review discusses current tissue engineering materials (e.g., growth factors and scaffolds) and methods (e.g., dynamic stretch and contact guidance) used to promote cellular synthesis and assembly of elastic matrix superstructures, and the limitations of these approaches when applied to smooth muscle cells, the primary elastin-generating cell type in vascular tissues. The potential application of these methods for in situ regeneration of disrupted elastic matrix at sites of proteolytic vascular disease (e.g., abdominal aortic aneurysms) is also discussed. Finally, the review describes the potential utility of alternative cell types to elastic tissue engineering and regenerative matrix repair. Future progress in the field is contingent on developing a thorough understanding of developmental elastogenesis and then mimicking the spatiotemporal changes in the cellular microenvironment that occur during that phase. This will enable us to tissue engineer clinically applicable elastic vascular tissue replacements and to develop elastogenic therapies to restore homeostasis in de-elasticized vessels.

Induced elastic matrix deposition within three-dimensional collagen scaffolds.

The structural stability of a cyclically distending elastic artery and the healthy functioning of vascular smooth muscle cells (SMCs) within are maintained by the presence of an intact elastic matrix and its principal protein, elastin. The accelerated degradation of the elastic matrix, which occurs in several vascular diseases, coupled with the poor ability of adult SMCs to regenerate lost elastin, can therefore adversely impact vascular homeostasis. Similarly, efforts to tissue engineer elastic matrix structures are constrained by our inability to induce adult cells to synthesize tropoelastin precursors and to crosslink them into architectural mimics of native elastic matrices, especially within engineered constructs where SMCs/fibroblasts primarily deposit collagen in abundance. In this study, we have shown that transforming growth factor-beta1 (TGF-ß1) and hyaluronan oligomers (HA-o) synergistically enhance elastic matrix deposition by adult rat aortic SMCs (RASMCs) seeded within nonelastogenic, statically loaded three-dimensional gels, composed of nonelastogenic type-I collagen. While there was no substantial increase in production of tropoelastin within experimental cases compared to the nonadditive control cultures over 3 weeks, we observed significant increases in matrix elastin deposition; soluble matrix elastin in constructs that received the lowest doses of TGF-ß1 with respective doses of HA-o, and insoluble matrix at the highest doses that corresponded with elevated lysyl-oxidase protein quantities. However, despite elastogenic induction, overall matrix yields remained poor in all experimental cases. At all provided doses, the factors reduced the production of matrix metalloproteinases (MMP)-9, especially the active enzyme, though MMP-2 levels were lowered only in constructs cultured with the higher doses of TGF-ß1. Immuno-fluorescence showed elastic fibers within the collagen constructs to be discontinuous, except at the edges of the constructs. Von Kossa staining revealed no calcific deposits in any of the cases. This study confirms the benefits of utilizing TGF-ß1 and HA-o in inducing matrix elastin synthesis by adult RASMCs over nonadditive controls, within a collagenous environment, that is not inherently conducive to elastogenesis.