Collagenous tissue engineered constructs to investigate the role of decorin on collagen fibrillogenesis and tissue mechanics
Grande-Allen, K. Jane
Doctor of Philosophy
Decorin, a small leucine rich proteoglycan, has been reported to control collagen fibrillogenesis, thereby influencing the tensile properties of collagenous tissues. In this research work, decorin deficient cells (Dcn -/- ) were combined with tissue-engineered collagen gels to study the contribution of decorin to cell proliferation, collagen fibrillogenesis, and tensile strength, as well as the interaction between decorin and transforming growth factor (TGF)-beta. This project was driven by the hypothesis that decorin deficient cells grown in tissue-engineered constructs would show significant biomechanical influence over the tensile behavior of collagenous tissues by controlling collagen fibrillogenesis. Since decorin inhibits cell adhesion and cell migration on various matrix molecules, such as collagen and fibronectin, the adhesive characteristics of Dcn -/- cells were first investigated to understand how the absence of decorin would influence cell organization in the collagen gels. Since β 1 integrins play an integral role in cell-mediated adhesion, the contributions of α 2 and β 1 subunits of integrin were also investigated. In this study, Dcn -/- cells showed significantly higher adhesion to both collagen and fibronectin substrates. For both collagen and fibronectin substrates, blocking either α 2 or β 1 integrin subunits blocked cell adhesion differently for the wild-type and Dcn -/- cells, suggesting that distinct mechanisms of adhesion are utilized by these cell types. Finally, Dcn -/- cells showed greater migration on the collagen substrate compared to wild-type controls. To further determine how decorin participates in matrix organization, collagen gels containing the Dcn -/- cells and wild-type control cells were grown under 2 different mechanical conditions—static tension and dynamic tension. The static tension collagen gels seeded with Dcn -/- cells showed greater gel contraction, matrix organization, ultimate tensile strength and elastic modulus than those seeded with wild-type cells. Moreover, addition of TGF-beta to the wild-type cell-seeded gels made them similar to Dcn -/- cell-seeded gels. Conversely, when the collagen gels containing Dcn -/- cells were treated with a TGF-beta receptor kinase inhibitor, they demonstrated reduced contraction. These results indicate that the inhibitory interaction between decorin and TGF-beta significantly influenced the matrix organization and material behavior of these in vitro model tissues. The dynamic gels were grown with the above cell types in a Flexcell Tissue Train™ culture system under cyclic 5% uniaxial strain at 0.1 Hz, while the static gels were cultured under static tension. Interestingly, it was found that some measured outcomes such as collagen fibril density, PG density, maximum load and stiffness were altered with mechanical stimulation regardless of the cell type used. On the other hand, unique outcomes regarding cell density, collagen fibril diameter, and biglycan expression were observed in response to cyclic strain in the Dcn -/- cell-seeded gels only. These results led us to conclude that decorinmediated tissue organization is heavily dependent upon tissue type and the amount of strain imparted on the tissue.
Biomedical research; Applied sciences; Collagenous tissue; Decorin Fibrillogenesis; TGF-beta Tissue engineering