Effects of the mechanical properties of collagen gel on the in vitro formation of microvessel networks by endothelial cells.

Tissue Engineering. 2007 Jul;13(7):1443-53.

Note: Figure captions are directly from the paper.

Introduction:

In recent years, tissue-engineered organs have become an increasingly promising source of organs for organ replacement therapy. Like those naturally found in the body, tissue-engineered organs require vascular networks to provide cells with nutrients and oxygen, as well as to remove waste products. However, the process by which these networks are constructed is not well-characterized, and thus, tissue-engineered organs cannot yet be vascularized reliably in the laboratory.

Recent studies suggest that the formation of vascular networks is heavily dependent upon the interaction between endothelial cells (ECs) and the surrounding extracellular matrix (ECM). In particular, in vitro models suggest that the mechanical properties of the ECM substrate govern many important cellular processes, including proliferation, migration, and differentiation. Although these effects have been observed, direct correlations between specific mechanical properties and cellular responses have not yet been established, and thus, these phenomena have not been understood on any quantitative level.

Summary:

General summary

The purpose of this study was to understand the effect that the stiffness of a substrate has on the formation of three-dimensional vasculature. To characterize this effect, bovine pulmonary microvascular ECs (BPMECs) were grown on collagen gels of varying stiffness in the presence of an angiogenic factor. Several different forms of microscopy (light, confocal, scanning electron, and transmission electron microscopy) were used to characterize the cellular response to the stiffness of the growth matrix.

Summary of results

Collagen gel stiffness is modulated by changing the pH of the gel

A series of collagen gels were prepared and polymerized under pH conditions between 5 and 10. The mechanical properties of these gels were characterized through uniaxial compression tests, from which stress-strain curves were derived. Based upon these curves, a relaxation modulus (a parameter for assessing the stiffness of viscoelastic materials) was calculated for each of the gels.

Figure 1 shows a plot of the relaxation modulus as a function of the pH of the gel. As indicated in the graph, the stiffness parameter appears to be linearly correlated with the pH of the gel, and there was roughly a 5-fold increase in relaxation modulus for an increase in pH from 5 to 9. In each of the subsequent cell studies, a gel at pH 5 was termed “flexible,” while gels produced at a pH of 9 were “rigid”.

Collagen gel stiffness affects the structure of cellular 3D networks

Using confocal laser scanning microscopy, the three-dimensional structures of the capillary-like networks formed by the cells were observed. Figure 3 (as numbered in the paper) shows the networks that were produced in flexible (A, B) and rigid (C, D) gels. As the paper discusses, “In the flexible gel, most cells…formed thin, dense networks,” while the rigid gels seemed to encourage the formation of “thick networks that were sparsely distributed” (Yamamura, et al., 2010). In addition, the networks formed in the flexible gel were found to be relatively shallow, primarily distributed 0-50 mm below the gel surface. By contrast, networks in the rigid gels reached significantly deeper (up to 120 mm below the gel surface).

Discussion and comments:

The purpose of this investigation was to consider the effect of collagen gel stiffness on the formation of three-dimensional vasculature by endothelial cells. This was accomplished through the use of an in vitro 3-D angiogenesis model coupled with several different forms of microscopy. In combination, these techniques indicated that BPMECs produce dramatically different vascular networks in response to differences in matrix stiffness.

Although this study suggests that matrix stiffness changes the cellular response, it provides no indication as to the molecular basis for this difference. Indeed, the discussion concludes, “This difference [in network morphology] may be a result of differences in cell-ECM interactions…” Of course, this correlation was discussed in broad terms in the introduction, and this statement suggests that no further light was shed on the cellular response through this study.

The authors of this study appear to have produced a novel 3-D angiogenesis model for studying the formation of vascular networks. In particular, this investigation shows that the mechanical properties of a collagen gel can be modulated by varying the pH conditions under which the gel is produced. It is unclear as to whether this is a standard protocol, but it does not appear to be a precise one. In particular, the standard deviations in relaxation modulus at each of the pH values (Figure 1) are quite significant, even averaged over nearly thirty gels. This result suggests that the stiffness of the gel cannot be controlled reproducibly to produce quantitative results. Thus, a different model may be required to study the effect of matrix stiffness on the morphology of vascular networks.