Confined compression of a tissue-equivalent: collagen fibril and cell alignment in response to anisotropic strain.

A method to impose and measure a one dimensional strain field via confined compression of a tissue-equivalent and measure the resulting cell and collagen fibril alignment was developed Strain was determined locally by the displacement of polystyrene beads dispersed and entrapped within the network of collagen fibrils along with the cells, and it was correlated to the spatial variation of collagen network birefringence and concentration. Alignment of fibroblasts and smooth muscle cells was determined based on the long axis of elongated cells. Cell and collagen network alignment were observed normal to the direction of compression after a step strain and increased monotonically up to 50% strain. These results were independent of time after straining over 24 hr despite continued cell motility after responding instantly to the step strain with a change in alignment by deforming/convecting with the strained network. Since the time course of cell alignment followed that of strain and not stress which, due to the viscoelastic fluid-like nature of the network relaxes completely within the observation period, these results imply cell alignment in a compacting tissue-equivalent is due to fibril alignment associated with anisotropic network strain. Estimation of a contact guidance sensitivity parameter indicates that both cell types align to a greater extent than the surrounding fibrils.

Mechanisms of stiffening and strengthening in media-equivalents fabricated using glycation.

We have recently reported that glycation can be exploited to increase the circumferential tensile stiffness and ultimate tensile strength of media-equivalents (MEs) and increase their resistance to collagenolytic degradation, all without loss of cell viability (Girton et al., 1999). The glycated MEs were fabricated by entrapping high passage adult rat aorta SMCs in collagen gel made from pepsin-digested bovine dermal collagen, and incubated for up to 10 weeks in complete medium with 30 mM ribose added. We report here on experiments showing that ME compaction due to traction exerted by the SMCs with consequent alignment of collagen fibrils was necessary to realize the glycation-mediated stiffening and strengthening, but that synthesis of extracellular matrix constituents by these cells likely contributed little, even when 50 micrograms/ml ascorbate was added to the medium. These glycated MEs exhibited a compliance similar to arteries, but possessed less tensile strength and much less burst strength. MEs fabricated with low rather than high passage adult rat aorta SMCs possessed almost ten times greater tensile strength, suggesting that alternative SMCs sources and biopolymer gels may yield sufficient strength by compositional remodeling prior to implantation in addition to the structural remodeling (i.e., circumferential alignment) already obtained.

Exploiting glycation to stiffen and strengthen tissue equivalents for tissue engineering.

Glycation, the nonenzymatic crosslinking of proteins by reducing sugars, is known to cause stiffening of soft tissues over a lifetime, particularly in diabetics. We show here that glycation due to elevated glucose and ribose concentrations in cell culture medium can be exploited in a matter of a few weeks of incubation to stiffen and strengthen tissue equivalents and to increase their resistance to collagenolytic degradation, all without loss of cell viability. Glycated tissue equivalents did not elicit inflammation or induce calcification upon subcutaneous implantation; rather, they were permissive to host integration and remodeling. Thus a pathological process might be used in a targeted way in tissue engineering to fabricate tissue equivalents with the required mechanical properties and desired resorption rate upon implantation.

Magnetic-induced alignment of collagen fibrils in tissue equivalents.

The use of reconstituted type I collagen gel as a scaffold for engineered soft tissues is a highly attractive prospect, given that collagen is the principal component of the extracellular matrix (ECM) in vivo, providing a mechanically suitable and information-rich scaffold for cell-ECM interactions. It has the advantage that cells can be directly entrapped within the comprising collagen fibrils as they grow into an entangled network from a cell containing solution of monomeric type I collagen. These tissue equivalents have the further advantage that the collagen fibrils can be aligned by applying a magnetic field during fibrillogenesis. Then, through a process termed "contact guidance," the cells align with the fibrils by directing their motility. Such alignment is characteristic of many tissues, and may provide microstructural and mechanical cues for regulation of cell phenotype and function, as well as influence the gross mechanical properties of the tissues. Recent research in our laboratory has used magnetic-induced alignment in the fabrication of tissue-equivalents, notably circumferential alignment in tubes, and longitudinal alignment in rods (patent pending). The former is aimed at reproducing the architecture of the arterial media; the latter is aimed at providing a bridge that promotes directed axonal growth between severed nerve ends. These tissue engineering applications exploit the finding of Torbet and Ronziere (1) in their cell-free studies that forming fibrils tend to align in the plane normal to the direction of the field (because of the negative diamagnetic anisotropy of collagen) and parallel to the mold surfaces (because of an uncharacterized interfacial effect).

Engineered alignment in media equivalents: magnetic prealignment and mandrel compaction.

We predicted and measured the evolution of smooth muscle cell (SMC) orientation in media-equivalents (MEs) for four fabrication conditions (F-, M-, F+, M+) under Free or Mandrel compaction (F/M) with and without magnetic prealignment of the collagen fibrils in the circumferential direction (+/-). Mandrel compaction refers to SMC-induced compaction of the ME that is constrained by having a nonadhesive mandrel placed in the ME lumen. Predictions were made using our anisotropic biphasic theory (ABT) for tissue-equivalent mechanics. Successful prediction of trends of the SMC orientation data for all four fabrication cases was obtained: maintenance of the initial isotropic state for F-, loss of initial circumferential alignment for F+, development of circumferential alignment for M-, and enhancement of initial circumferential alignment for M+. These results suggest two mechanisms by which the presence of the mandrel leads to much greater mechanical stiffness in the circumferential direction reported for mandrel compacted MEs relative to free compacted MEs: (1) by inducing an increasing circumferential alignment of the SMC and collagen, and (2) by inducing a large stress on the SMC, resulting in secretion and accumulation of stiffening components.

Magnetically orientated tissue-equivalent tubes: application to a circumferentially orientated media-equivalent.

Circumferential orientation of collagen fibrils in a media-equivalent (ME) is achieved in a simple and effective way using the orientating effects of a strong magnetic field during collagen fibrillogenesis when the ME is first created. Circumferential orientation of the entrapped smooth muscle cells (SMC) is achieved subsequently via cell contact guidance, the induced SMC orientation along orientated fibrils. After describing the methods used, several lines of evidence are provided showing that the magnetically orientated ME is circumferentially orientated, including collagen birefringence, circumferential SMC orientation, accelerated ME compaction and increased ME stiffness with reduced creep in the circumferential direction as compared to control MEs not exposed to a magnetic field during fibrillogenesis. The optimization of these methods is discussed in order to better mimic the circumferential orientation and mechanical properties of a natural medium. Other applications of magnetically orientated tissue-equivalents are indicated.