Effects of mechanical stretch on collagen and cross-linking in engineered blood vessels

Abstract

It has been shown that mechanical stimulation affects the physical properties of multiple types of engineered tissues. However, the optimum regimen for applying cyclic radial stretch to engineered arteries is not well understood. To this end, the effect of mechanical stretch on the development of engineered blood vessels was analyzed in constructs grown from porcine vascular smooth muscle cells. Cyclic radial distension was applied during vessel culture at three rates: 0 beats per minute (bpm), 90 bpm, and 165 bpm. At the end of the 7-week culture period, harvested vessels were analyzed with respect to physical characteristics. Importantly, mechanical stretch at 165 bpm resulted in a significant increase in rupture strength in engineered constructs over nonstretched controls. Stress-strain data and maximal elastic moduli from vessels grown at the three stretch rates indicate enhanced physical properties with increasing pulse rate. In order to investigate the role of collagen cross-linking in the improved mechanical characteristics, collagen cross-link density was quantified by HPLC. Vessels grown with mechanical stretch had somewhat more collagen and higher burst pressures than nonpulsed control vessels. Pulsation did not increase collagen cross-link density. Thus, increased wall thickness and somewhat elevated collagen concentrations, but not collagen cross-link density, appeared to be responsible for increased burst strength.

Figure 1

Histologies of engineered vessels. Vessels were grown at three radial distension rates: 0 beats per minute (bpm; nonstretched control), 90 bpm, and 165 bpm. (A, D) Nonstretched control vessels; (B, E) vessels cultured at 90 bpm; (C, F) vessels cultured at 165 bpm. (A–C) H&E stains; (D–F) Masson's trichrome, where collagen stains blue, cell bodies stain red, and glycosaminoglycans stain green. PGA polymer fragments more noticeable as oval or rectangular objects (D–F), staining blue. Wall thickness is increased in pulsed vessels. Scale bars: 50 μm.

Figure 2

Burst pressure measurements of engineered porcine vessels. Rupture strengths were measured from engineered vessels harvested at the end of culture. Vessels were grown at three radial distension rates: 0 beats per minute (bpm; nonpulsed control; n = 6), 90 bpm (n = 7), and 165 bpm (n = 8). Error bars represent SEM. *Statistically significant differences in comparing average burst pressures of engineered vessels grown at 165 bpm compared to nonstretched controls (p < 0.05).

Figure 3

Representative stress–strain curves of engineered porcine vessels. Representative stress–strain curves for vessels grown with mechanical stimulation at 0 bpm (squares), 90 bpm (triangles), and 165 bpm (circles).

Figure 4

Maximal elastic modulus for engineered porcine vessels. Maximum moduli were measured from engineered vessels harvested at the end of culture. Vessels were grown at three radial distension rates: 0 beats per minute (bpm; nonpulsed control; n = 6), 90 bpm (n = 4), and 165 bpm (n = 8). Error bars represent SEM.

Figure 5

Collagen and cross-link densities measured in engineered porcine vessels. Collagen (A) and cross-link densities (B) were measured in engineered vessels at the end of culture using hydroxyproline assay and HPLC, respectively. Vessels were grown at three radial distension rates: 0 beats per minute (bpm; nonpulsed control; n = 4), 90 bpm (n = 6), and 165 bpm (n = 8). Error bars represent SEM. While the trend in collagen accumulation in response to pulsation mirrors observations regarding mechanics, the trend in cross-link formation does not. HP, hydroxylysylpyridinoline.