Induced Elastic Matrix Deposition Within Three-Dimensional Collagen Scaffolds

Abstract

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-b1) 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-b1 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-b1. 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-b1 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.

Impact of Cyclic Stretch on Induced Elastogenesis Within Collagenous Conduits

“…The addition of EFs by themselves impacted SMC elastogenesis. Similar to our previous study with rat SMCs, 42 this study with human SMCs did not show any differences in cell densities after 21 days of treatment with a low dose of EFs (i.e., 0.1 ng/mL TGF-b1 and 0.2 mg/mL HA-o). While we did find evidence of the elastogenic benefit of EFs relative to static, no-EF controls, it was not as dramatic as we had previously observed in the static model.…”

“…This can be attributed to possible limitations to in-diffusion and bioavailability of exogenous EFs to cells within the 3D constructs. While higher doses of the factors, and of TGF-b in particular, have been shown to attenuate SMC proliferation, 42 even the low dose used in this study did not impact this. At the tested doses, the EFs also increased tropoelastin gene expression more than fourfold for static cultures, although the increases in alkali-soluble elastic matrix content were not statistically significant.…”

“…14 We also showed that cell alignment within rectangular constructs maintained under static uniaxial stretch occurs in the direction of applied strain, positively influencing orientation and alignment of both the preexisting collagen matrix and the cell-synthesized elastic matrix.…”

“…This result was surprising since our previous study with rectangular collagen constructs showed that the EFs significantly enhance matrix elastin deposition, particularly the alkali-soluble matrix elastin. 42 These differences could be due to the fact that human SMCs are induced to a lesser extent by EFs than rat SMCs. 30 This result supports the need to provide additional stimuli, such as cyclic stretch, to further improve elastic matrix generation by human SMCs.…”

“…Elastogenic factors (EFs) were also added to stimulate cellular elastic matrix generation in this study, with the doses (i.e., 0.1 ng/mL transforming growth factor-beta 1 [TGF-b1] and 0.2 mg/mL hyaluronan oligomers [HA-o]) based on results obtained previously. 14 In light of results of other studies in our lab that demonstrate that human aortic smooth muscle cells (HASMCs) respond to elastogenic stimulation by EFs, 30 we have focused on study of this cell type to provide greater clinical relevance.…”