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-β1) 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-β1 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-β1. 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-β1 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.

FIG. 1.

Statically loaded rat aortic smooth muscle cell-seeded three-dimensional collagen gels. Gels, shown at day 1 of culture, were cast within silicone wells and anchored to polyurethane end-holders. Color images available online at www.liebertonline.com/tea

FIG. 2.

Percentage change in central widths of constructs at day 21 compared to day 0 (n=6 per case). *Significance of differences compared to control cultures, deemed for p<0.05. ^#Significance of differences in outcomes between treatments paired by horizontal bars, deemed for p<0.05.

FIG. 3.

Effect of elastogenic factors on cell proliferation ratio. Cell numbers were calculated based on the estimate of 6 pg of DNA per cell, as calculated from DNA assay, at 0 and 21 days postseeding, and their ratios determined and compared to controls (n=6 replicate constructs per treatment). Differences were deemed to be significant from control cultures for p<0.05 (*). ^#Significance of differences in outcomes between treatments paired by horizontal bars, deemed for p<0.05.

FIG. 4.

Effect of TGF-β1 and HA-o on elastin synthesis. (A) Differences in synthesis of tropoelastin and total matrix elastin per construct in samples cultured with HA-o and TGF-β1 relative to control cultures. (B) Effect of factor treatment on synthesis of alkali-soluble matrix elastin and crosslinked, alkali-insoluble matrix elastin. (C) Effect of factor treatment on yields of matrix elastin, that is, the fraction of total elastin amounts produced by cells that is deposited in the matrix. *Differences from controls significant for p<0.05 (n=6 for each case). ^#Significance of differences in outcomes between treatments paired by horizontal bars, deemed for p<0.05. TGF-β1, transforming growth factor-beta1; HA-o, hyaluronan oligomer.

FIG. 5.

Effect of factors on enzyme synthesis and active forms. Representative immunoblots of (A) MMP-9 and (B) MMP-2 indicating the active and inactive protein bands for each case, and (C) LOX. (D) Representative gelatin zymogram of MMP-2 indicating the active enzyme band at 62 kDa and zymogen at 72 kDa. LOX, lysyl oxidase; MMP, matrix metalloproteinase.

FIG. 6.

Differences in total protein synthesis of MMP-2 and −9 in experimental cases compared to control, as determined from immunoblotting of the two proteases. *Statistical difference compared to control for p<0.05 (n=3 per case). ^#Significance of differences in outcomes between treatments paired by horizontal bars, deemed for p<0.05c (n=3 per case).

FIG. 7.

Effect of factors on MMP-2 quantities as determined by gelatin zymography. Data represent a fold change in relative density units of zymogram bands in experimental cases compared to control cultures. *Significance in differences compared to control for p<0.05 (n=3 per case). ^#Significant differences in MMP quantities (p<0.05) between pairs of treatment groups indicated by the horizontal lines.

FIG. 8.

Fold change in protein levels of LOX in cultures with TGF-β1 and HA-o factors compared to untreated control cultures, as determined by western blotting. *Differences from control were deemed significant for p<0.05 (n=3 per case). ^#Significance of differences in outcomes between treatments paired by horizontal bars, deemed for p<0.05.

FIG. 9.

Representative images of 5 μm sections stained for elastin with modified Verhoeff Van Gieson at 20×magnification (scale bar=200 μm). Elastic fibers are stained purple and surrounding collagen is stained pink. Color images available online at www.liebertonline.com/tea

FIG. 10.

Representative fluorescence micrographs of construct sections showing distribution of nuclei in green (A), elastin/elastic fibers in red (B), and the overlay of the two (C). Paraffin-embedded sections were treated with Pontamine sky blue to quench autofluorescence of collagen and shift that of elastin to the red wavelengths. Nuclei were stained with DAPI. Magnification: 20×(scale bar=100 μm). Color images available online at www.liebertonline.com/tea

FIG. 11.

Elastic fiber alignment in collagen gel constructs represented by angular standard deviation in degrees (A) and number of aligned fibers (B). *Significance in difference from nonfactor-treated control cultures, deemed for p<0.05. ^#Significance of differences in outcomes between treatments paired by horizontal bars, deemed for p<0.05.

FIG. 12.

Von Kossa staining for mineral deposits. The absence of any black spots/deposits indicates lack of TGF-β1-induced matrix mineralization in the dose range studied. Aneurysmal rat aortae were stained as positive controls and show the presence of sub-intimal calcific deposits (black). Cell nuclei are stained pink in all cases. Scale bar=500 μm. Color images available online at www.liebertonline.com/tea