A dynamic straining bioreactor for collagen-based tissue engineering

Abstract

Collagen-based tissue constructs for vascular, orthopedic, and other applications have been studied extensively for many years (L'Heureux et al., 1993; Seliktar et al., 2000; Shi et al., 2002; Shi and Vesely, 2004; Shi and Vesely, 2003; Tranquillo et al., 1996; Weinberg and Bell, 1986). These models involve first mixing soluble, fibrillar collagen with the appropriate cells, serum and medium. After the collagencell mixture is neutralized and brought up to 37°C, soluble collagen reassembles into fibrils and a gel is created. Cells become entrapped within the collagen gel and begin to interact with the collagen fibrils. These cells reorganize the surrounding collagen matrix, contract it and exclude water. In many ways, this in-vitro contraction mimics wound healing in vivo (Grinnell, 1994). When the gel is mechanically constrained, the collagen fibrils align in the direction of constraint, and a highly aligned, compacted collagenous construct can thus be fabricated. The collagen fibrils can therefore be manipulated into useful structures using the principle of directed collagen gel shrinkage. So far, inappropriate mechanical properties have been one of the main limitations of most collagen-based tissue equivalents (Girton et al., 1999; Kanda and Matsuda, 1994; Kanda et al., 1993a; Kanda et al., 1993b; Kim et al., 1999; Seliktar, et al., 2000; Tranquillo et al., 1992). Various methods for improving these constructs have been developed and evaluated. They include mandrel compaction (L'Heureux, et al., 1993), magnetic prealignment (Tranquillo, et al., 1996), glycation etc (Girton, et al., 1999). Mechanical stimuli are known to exert a variety of effects on smooth muscle cells (SMCs) in culture, increasing proliferation, matrix production and expression of phenotype-specific proteins and growth factors (Keeley and Bartoszewicz, 1995; O'Callaghan and Williams, 2000; Seliktar et al., 2001; Stanley et al., 2000; Williams, 1998; Wilson et al., 1995). It is also known that fibroblasts can generate substantial traction forces in culture that are, in part, regulated by external mechanical forces (Akhouayri et al., 1999; Brown et al., 1998; Brown et al., 1996; Fredberg et al., 1997; Petroll et al., 1993; Shelburne and Pandy, 1997). The contractile forces generated by fibroblasts in three-dimensional collagen substrates have been studied for many years (Arora et al., 1999; Brown, et al., 1998; Brown et al., 2002). The observation that external mechanical forces can regulate both fibroblast contractile forces and matrix production suggested to us that mechanically stimulating the constructs could improve their microstructure and enhance their mechanics. The enhanced cell growth and extracellular matrix (ECM) production, as well as the improved structural integrity of collagen constructs, indeed appear to be related directly to the application of cyclic strain. Our application of a dynamic loading bioreactor is for the fabrication of tissue engineered mitral valve chordae. These "one dimensional" structures need to be loaded only longitudinally, requiring a relatively simple dynamic bioreactor. Dynamic mechanical conditioning systems being used by most groups make use of compressed air. We were interested in developing a much simpler, very inexpensive system, and thus focused on a cam-based approach. We also used stereolithography (SLA) for the fabrication of the dynamic bioreactor. SLA is typically used for rapid prototyping, not for fabrication of the final product. We found that the resin from which SLA components are fabricated withstands autoclaving, is not toxic to cells (once thoroughly washed), and is very cost effective. It can thus be used for large-scale product manufacturing and may help offset some of the huge scale-up costs that have plagued the tissue engineering industry. © 2005 Springer.