Stabilized collagen scaffolds for heart valve tissue engineering

Abstract

Scaffolds for heart valve tissue engineering must function immediately after implantation but also need to tolerate cell infiltration and gradual remodeling. We hypothesized that moderately cross-linked collagen scaffolds would fulfill these requirements. To test our hypothesis, scaffolds prepared from decellularized porcine pericardium were treated with penta-galloyl glucose (PGG), a collagen-binding polyphenol, and tested for biodegradation, biaxial mechanical properties, and in vivo biocompatibility. For controls, we used un-cross-linked scaffolds and glutaraldehyde-treated scaffolds. Results confirmed complete pericardium decellularization and the ability of scaffolds to encourage fibroblast chemotaxis and to aid in creation of anatomically correct valve-shaped constructs. Glutaraldehyde cross-linking fully stabilized collagen but did not allow for tissue remodeling and calcified when implanted subdermally in rats. PGG-treated collagen was initially resistant to collagenase and then degraded gradually, indicating partial stabilization. Moreover, PGG-treated pericardium exhibited excellent biaxial mechanical properties, did not calcify in vivo, and supported infiltration by host fibroblasts and subsequent matrix remodeling. In conclusion, PGG-treated acellular pericardium is a promising scaffold for heart valve tissue engineering.

FIG. 1.

Characteristics of acellular porcine collagen scaffolds. (a) Hematoxylin and eosin staining of acellular scaffold showing wavy collagen fibers and complete lack of cell staining, compared with native tissue (b) in which cells are outlined by black arrows. (c) Verhoeff van Gieson staining showing fine elastin fibers (black arrow) throughout the native pericardial tissue, which were completely removed (d) by elastase treatment. (e) Presence of detergent-soluble proteins in fresh pericardium and acellular collagen scaffolds was evaluated using electrophoresis and silver staining. (f) Agarose and ethidium bromide gel electrophoresis of DNA extracted from fresh pericardium and the acellular collagen scaffolds; (g) densitometry of DNA bands in (f). (h) Enzyme-linked immunosorbent assay for matrix metalloproteinase 2 in fresh pericardium and the acellular collagen scaffolds, (i) chemotaxis of fibroblasts toward collagen peptides obtained from acellular pericardium; inset in (i) shows a cell undergoing mitosis after chemotaxis, (j) positive chemotaxis control, (k) negative control. Color images available online at www.liebertonline.com/ten.

FIG. 2.

(a) Resistance to collagenase of treated collagen scaffolds: a time-course study. Percentage mass loss after collagenase treatment is shown for untreated scaffolds (control) and for glutaraldehyde and penta-galloyl glucose (PGG)-treated scaffolds. Control values were all significantly higher than other groups. Analysis of variance statistical significance between groups. (b) Chemical structure and three-dimensional model of the PGG molecule. The central derivatized glucose core (blue) and the external gallic acid residues (pink circles) are depicted at left. Atoms in the three-dimensional model are C = black, O = red, H = light gray. Color images available online at www.liebertonline.com/ten.

FIG. 3.

(a) Representative equibiaxial tension protocol for acellular collagen scaffolds. (b) Peak stretch ratios along the fiber-preferred direction and cross-fiber-preferred direction for control untreated scaffolds and for glutaraldehyde and penta-galloyl glucose–treated scaffolds. (c) Hysteresis values calculated for the four scaffold groups. (d) Comparison of axial coupling of fiber-preferred direction for the four scaffold groups. *Analysis of variance statistical significance between groups.

FIG. 4.

Histological analysis of subdermally implanted scaffolds. Representative hematoxylin and eosin micrographs from 3-week samples showing (a) pre-implant collagen scaffolds, (b) explanted control scaffolds, (c) explanted glutaraldehyde-treated scaffolds, (d) explanted penta-galloyl glucose (PGG)-treated scaffolds. Black arrows point to cells present within implants. (f) Phenol staining showing collagen-bound PGG (brown) before implantation and (g) 3 weeks after subdermal implantation. Inset in (f) represents phenol staining of a negative control (scaffold not treated with PGG). Color images available online at www.liebertonline.com/ten.

FIG. 5.

Immunohistochemical identification of infiltrating cells. (a) Vimentin stain showing extensive fibroblast-like infiltrating cells in all implant groups at 1 week (1w), 3 weeks (3w), and 6 weeks (6w) after implantation. (b) Proline-hydroxylase immunohistochemical staining; arrowheads point to positively stained cells. (c) Specific staining showing few macrophages (arrowheads). Inset in (c) shows macrophage staining of rat spleen sections used as a positive control. Sections were counterstained with hematoxylin (nuclei dark blue). Bars are 50 μm in all micrographs. Color images available online at www.liebertonline.com/ten.

FIG. 6.

(a) Matrix metalloproteinase (MMP) activities in explanted scaffolds. Densitometry of active MMP bands are expressed as relative density units normalized to protein content. MMP-9 (a) and MMP-2 (b) were identified from their relative migration on gelatin containing 10% polyacrylamide gels (insets show representative zymograms). (b) Anatomically correct heart valve scaffolds. Silicone molds of porcine aortic valves were made by casting. (1) Note a portion of the ascending aorta, the emergence of the two coronary arteries (RC, LC) and the three cusps (A, B, C), which closely mimic valve anatomy. Decellularized porcine pericardium scaffolds were modeled into heart valve–shaped constructs using the molds and penta-galloyl glucose treated to cross-link and maintain shape (2) followed by immersion in saline (3). The position of the three cusps is marked by A, B, C. Color images available online at www.liebertonline.com/ten.