It is well known that cell growth and functionality is guided by the substrate. In mature arteries, the collagen network plays a structural and signalling role in the physiological response of SMC[56]. Scaffolds derived from natural biomaterials such as collagen have intrinsic cell adhesion properties[48]. Arterial SMC have very different characteristics when grown in culture than when found in normal vessels. The normal contractile phenotype changes to a more proliferating, protein secreting mode when grown in culture[61]. The morphology becomes less elongated, proliferation increases with each passage in culture and the expression of smooth muscle actin (SMA) expression decreases with each passage[62]. The factors that play a role in this phenotype change include the form of the substrate, seeding density, the presence of ECM proteins and growth factors, mechanical conditioning and the presence of endothelial cells.
The physical form of ECM molecules strongly influences gene expression of adherent cells[48]. In collagen gels, SMC appear much more elongated, appear to be more in a
contractile state, and therefore grow more slowly than those grown on plastic or on two- dimensional collagen sheets. SMA expression of SMC is also down regulated in collagen gels. Cells seeded below confluence maintain a synthetic phenotype until confluence is reached[63].
The ECM is known to affect cell function through both biochemical and mechanical signalling pathways. Culture of SMC on different ECM molecules affects cell phenotype by modulating morphology, proliferation and protein expression[62]. Collagen type IV, elastin and laminin maintain cells in the contractile phenotype while fibronectin promotes a change to a more synthetic phenotype[64,63].
Phenotype control can also be modulated by exogenous biochemical stimulation such as growth factors. Growth factors are proteins which promote proliferation and migration of cells via interactions with specific cell-membrane receptors. To this effect, ascorbic acid, platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β) and heparin have been studied[65,62]. Table 1-2 shows an overall picture of the various growth factors and their effects. SMC and FC reach confluence faster in the presence of ascorbic acid[65]. In gels, PDGF caused an increase in cell number and a decrease in SMA. These effects were the opposite with cultures in two-dimensions. Although TGF-β causes an increase in SMA and a decrease in proliferation on flat surfaces, this factor was found to have little effect in three dimensions. Heparin slightly increases SMA in flat culture conditions but has no effect in gels. It does, however, decrease the growth rate of cells in gels and on flat surfaces. Therefore, the presence of a three-dimensional collagen matrix has an effect on cell morphology, proliferation and SMA expression. This matrix also affects the cells reactions to various biochemical signals. There is also a marked difference in SMA between cells grown in disc-shaped gels as compared to tubular gels compacted around a mandrel[42]. It is possible that the higher mechanical stresses in the latter case are sensed by the cells which react accordingly. This may also explain the varied morphological behaviour of SMC depending on their position in the disc-shaped gels and tubes[66]. In discs, cells on the surface of gels are more flattened. Cells on the bottom of gels are more organized into a cellular network than those in the middle which are more elongated. This
also applies to tubular gels in which the position of cells relative to the lumen may affect phenotype. Smooth muscle actin Proliferation Gel compaction Collagen synthesis Elastin synthesis Matrix metallo- proteinase synthesis PDGF (2D) ↑ ↓ N/A PDGF (3D) ↓ ↑ ↑ Heparin (2D) ↑ N/A Heparin (3D) − ↓ ↓ TGF-β (2D) ↑↑ ↓ N/A ↑ TGF-β (3D) − − ↑ ↑ Ascorbate ↑ ↓ Ascorbic Acid ↑ ↑ ↓ Insulin ↑
Insulin-like growth factor-1 ↑
Mechanical strain ↑ ↑ ↑ ↑
↑
↑
References [62] [74] [73] [60,71,94] [48] [42] [65]
Table 1-2 : The effects of various chemical signals on smooth muscle cell seeded collagen gels or films. (a blank box indicates no relevant influence was found in the literature.) Cyclic distension induces SMC to a more pronounced contractile phenotype[67]. It has been shown that SMC in the synthetic phenotype can revert to the contractile phenotype after implantation[68]. Shear stress also affects EC functionality. These mechanical environmental conditioning effects are discussed in more detail further on.
The engineering of a TEBV in this manner infers the co-culture of EC and SMC. This co- culture adds increased complexity than simply referring to two simple cultures of each cell type. EC and SMC can interact by two mechanisms: by body fluids and by direct contact[61]. EC secrete both inhibitors and stimulants of SMC growth and SMA expression such as TGF-β and PDGF. However, in co-culture, EC release more SMC growth inhibitory factors such as a heparin-related glycosaminoglycan and transforming growth factor β (TGF-β). Co-culture of these two cell types also results in completely different
endothelial properties. EC grown on SMC seeded collagen gels are more elongated than those grown in culture dishes.
It is also possible with the collagen gel method to culture all three vascular cell types together. It has even been demonstrated that it is unnecessary to separate SMC and FC prior to gel formation. Vessel replacement constructs with a homogeneous mixture of SMC and FC prior to implantation demonstrated a segregation of these two cell types after implantation with SMC accumulating on the subendothelial layer and FC accumulating on the outer layer[69].
Control over the phenotype of SMC is essential. Intimal hyperplasia caused by excessive growth of SMC at the TEBV-artery interface is a major impediment to long-term implantibility of TEBVs[18].
A major problem associated with TEBV without synthetic support is a lack of adequate mechanical integrity, more specifically stiffness, strength and elasticity. Collagen tubular constructs naturally stiffen during culture although this takes an extended period of time and may not be sufficient. Therefore, many approaches exist to attempt to improve mechanical properties of collagen vessel replacements. The main approaches to achieve this are discussed below.