Microaligned collagen matrices by hydrodynamic focusing: controlling the pH-induced self-assembly
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Microaligned collagen matrices by hydrodynamic focusing: Controlling the pH-induced self-assembly Sarah K¨oster1,2∗ , Jennie B. Leach2† , Joyce Y. Wong2 , and Thomas Pfohl1 1 Max Planck Institute for Dynamics and Self-Organization, G¨ottingen, Germany 2 Department of Biomedical Engineering, Boston University, Boston, MA ABSTRACT The hierarchical structure of type I collagen fibrils is a key contributor to the mechanical properties of the extracellular matrix (ECM). It is known that the process of in vitro fibrillogenesis strongly depends on the pH of the collagen solution. To date, there are few methods available for precisely controlling and investigating the dependence of collagen fibril assembly on the local pH. The objective of this work was to create highly defined pH gradients to systematically determine the effects of local pH on microscale collagen fibrillogenesis and alignment. We use a microfluidic mixing device to create a diffusion controlled pH gradient, which in turn initiates the self-assembly and concurrent flow-alignment of soluble collagen. Finite element method simulations of the hydrodynamic and diffusive phenomena are used to calculate the local concentrations of the components involved in the reaction. We develop a model to analytically calculate the local pH in the microfluidic device from these concentrations. A comparison with the experimental results from polarized light microscopy are in good agreement with the simulations. INTRODUCTION Collagen I is the most common type of all classified collageneous proteins. In contrast to other types which form networks, collagen I is a fibril-forming protein that self-assembles hierarchically at the nano-, micro-, and macroscales. It is mainly found in bone, skin and tendon but also in ligaments, the cornea and internal organs. Besides this noticeable biological importance collagen I plays a key role as a component for engineered functional tissue replacements [1-3]. While the pH dependence of collagen fibrillogenesis is known in principle [4,5], its understanding is complicated by the fact that existing methods cannot precisely predict the pH within the system. However, these pH conditions are crucial for the comprehension of the process of collagen assembly into its unique hierarchical organization. We are particularly interested in controlling structural properties such as the degree of alignment and at the same time investigating the dynamics of the assembly process in situ. Microfluidics has proven to be a powerful tool for the study of reactions on small length scales [6]. Here, we present a method which is used to orient and concurrently self-assemble collagen I. We use a cross geometry microfluidic diffusive mixing device containing three inlets and one outlet to align soluble collagen under hydrodynamic flow and initiate collagen self-assembly and fibril formation by creating a defined pH gradient in the outlet microchannel. Finite element method (FEM) simulations are used to model the conditions within the microchannels ∗
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