Regulating collagen assembly dynamics in vitro : an investigation from monomers to fibers.

• Online Access: http://hdl.handle.net/2047/D20413925

The composition, abundance, and organization of protein fibrils and fibers regulate the structure and mechanical performance of tissues and organs in the body. For example, unidirectional, or anisotropic, fibrous networks are present in tissues that endure tremendous forces and pressures, while isotropic networks are more commonly observed in tissues that experience little to no mechanical tension. While several efforts have been made to replicate tissue form and function in vitro through engineering efforts, the field remains challenged by (1) sourcing and handling the biomaterials in vitro under physiological conditions and (2) manipulating and polymerizing them without destroying important ultrastructural features. This work aims to address these challenges in the context of Type I collagen (COL), which is the primary extracellular matrix protein comprising all tissues. The overarching goals are to determine how other biomolecules regulate COL assembly dynamics (e.g. kinetics, thermodynamics, and organization) in vitro and to use this information together with existing and/or adapted manufacturing techniques to produce highly aligned COL scaffolds.First, we investigate the role of simple monosaccharides as a proxy for more complex, native glycosaminoglycans on regulating COL assembly dynamics. We show that increasing concentrations of monosaccharides slow then fully stop COL assembly and describe a potential mechanism behind this phenomenon based on solvent organization. In addition, we observe that the presence of monosaccharides does not degrade a pre-established COL network, and that soluble COL can still incorporate into a pre-existing network in spite of the nucleation-inhibiting conditions. Furthermore, we present a procedure for maintaining concentrations of monomeric COL three orders of magnitude above published threshold values at physiological conditions. Next, we explore the molecular interactions between COL and the glycoprotein fibronectin (FN) in vitro and examine whether fibrillar FN acts as a template to organize COL fibrils during polymerization. We describe a reciprocal relationship between FN and COL during fiber formation in vitro, where the polymerization of one catalyzes the assembly of the other. We then prototype methods for generating multiple FN fibers simultaneously to create templates for COL assembly. Lastly, we determine if the pre-assembled FN fibers have any influence on the alignment of COL fibrils during fibrillogenesis. Lastly, we design and develop a device that utilizes shear to simultaneously align COL fibrils as COL polymerizes, producing sheets of anisotropic fibrillar COL at the centimeter-scale. We analyze the effects of various parameters on fibril alignment such as shear rate, COL concentration, and degree of polymerization before exposure to shear. Furthermore, we investigate a potential mechanism behind the fibril alignment in the network. We then demonstrate that we can manipulate the dimensions and composition of the produced networks, highlighting the customizable nature of the material and its potential to play a role in a variety of applications. In summary, we have developed techniques for controlling both COL assembly dynamics and fibril organization, which could prove to be beneficial in the development of future tissue replicates and therapeutics.--Author's abstract