Initial scaffold thickness affects the emergence of a geometrical and mechanical equilibrium in engineered cardiovascular tissues

Abstract

In situ cardiovascular tissue-engineering can potentially address the shortcomings of the current replacement therapies, in particular, their inability to grow and remodel. In native tissues, it is widely accepted that physiological growth and remodelling occur to maintain a homeostatic mechanical state to conserve its function, regardless of changes in the mechanical environment. A similar homeostatic state should be reached for tissue-engineered (TE) prostheses to ensure proper functioning. For in situ tissue-engineering approaches obtaining such a state greatly relies on the initial scaffold design parameters. In this study, it is investigated if the simple scaffold design parameter initial thickness, influences the emergence of a mechanical and geometrical equilibrium state in in vitro TE constructs, which resemble thin cardiovascular tissues such as heart valves and arteries. Towards this end, two sample groups with different initial thicknesses of myofibroblast-seeded polycaprolactone-bisurea constructs were cultured for three weeks under dynamic loading conditions, while tracking geometrical and mechanical changes temporally using non-destructive ultrasound imaging. A mechanical equilibrium was reached in both groups, although at different magnitudes of the investigated mechanical quantities. Interestingly, a geometrically stable state was only established in the thicker constructs, while the thinner constructs' length continuously increased. This demonstrates that reaching geometrical and mechanical stability in TE constructs is highly dependent on functional scaffold design.

Keywords: cardiovascular; growth; remodelling; tissue-engineering.

Figure 1.

Schematic of the Vertigro bioreactor and pressure application system. Source: van Kelle et al. [36], Copyright: Mary Ann Liebert, granted permission for reprint.

Figure 2.

Geometrical changes of the constructs during dynamic culture: changes in tissue length L₀ (a) and construct thickness t₀ (b) for starting scaffold thicknesses of 0.31 mm (blue) and 0.47 mm (red). Significant changes (p < 0.05) between adjacent time points within the initially thinner and thicker constructs are indicated by * and $, respectively, while significant differences between the two groups at the same time point are indicated by #. Representative ultrasound images (with p = 0) of the thin (c,e) and thick (d,f) constructs at the start (c,d) and end (e,f) of the dynamic culturing.

Figure 3.

Assessment of the constructs’ mechanical state during dynamic culture. In all constructs, the stretch (a), maximum principal Cauchy stress (b), strain energy density (c) and tangent of the principal stress–stretch curve at p = 4 kPa (d) were non-destructively quantified. Significant changes (p < 0.05) between adjacent time points within the initially thinner and thicker samples are indicated by * and $, respectively, while significant differences between the two groups at the same time point are indicated by #.

Figure 4.

Mechanical testing of the bare scaffold. First Piola–Kirchhoff stress during fatigue testing with increasing actuator displacements (a). Images were captured before (c) and several hours after (d) fatigue testing, here shown for a sample of t₀ = 0.31 mm. The stress–stretch behaviour of each sample was characterized before (continuous lines) and after (dashed lines) fatigue testing (b).

Figure 5.

The top (right) and bottom (left) side of a representative collagen-stained quarter of one of the constructs. Zoomed-in images are indicated between the white squares, with below them scanning electron images of the scaffold prior to seeding. The histograms indicates the fraction of collagen fibres oriented in each direction for the entire sample.

Figure 6.

Representative histological images of the middle section for the thin (left column) and thick (right column) samples at t = 21 days of dynamic culture, where the bottom side of all samples coincides with the pressurized side in the bioreactor. (a,b) HE stain showing the general tissue composition, with a distinct layer formation on the top side of the samples. (c,d) PR stain showed general collagen organization (red). (e,f) PR stain visualized with polarized light, depicting relatively mature collagen fibres (red). (g,h) Fluorescent images with α-SMA-positive cells in green, cell nuclei in blue and collagen in red. (i,j) Fluorescent stain for collagen type III. (k,l) Co-fluorescent stain for elastin (red) and cell nuclei (blue). (m,n) SO stain depicting GAGs in red. The scale of the images is indicated by the black and white bars (100 µm) (HE, hematoxylin & eosin; PR, picosirius red; SO, safarin O).

Figure 7.

Biochemical assays: total DNA content (μgram) (a) GAGs per DNA (b) and HYP per DNA (c) for initial scaffold thicknesses of 0.31 mm (white bars) and 0.47 mm (black bars).