Investigating the effects of hypoxic culture and macromolecular crowding on engineered scaffold-free tendon fiber development

Abstract

Though functional tendon engineering has made great advances in the last several decades, many tendon engineering approaches are limited by prolonged culture times required for construct maturation. This thesis investigated the application of developmentally-inspired stimuli (i.e., macromolecular crowding, hypoxia) to enhance biomechanical maturation of engineered tendon, using our lab’s established scaffold-free tendon fiber engineering platform. Culture in low oxygen (5%) or media crowded with a polydisperse Ficoll cocktail improved biomechanical properties of fibers after only 1 day in culture. Interestingly, biomechanical properties diverged beyond this point, with hypoxic fibers strengthening linearly with time while more delayed biomechanical improvements were seen with crowding. Notably, as with cyclic tensile strain, no differences in extensibility or low-load behavior (i.e., toe-in strain) were seen with stimulation or time in culture. When assessing underlying changes in gene expression, hypoxic culture and crowding each upregulated gene expression of Collagen types 1 and 3 (Col-1 and Col-3), the major components of developing tendon, with further increases observed when combined. Hypoxia also upregulated lysyl oxidase (LOX) expression, an indicator of collagen crosslinking. Though prior work in our lab observed an upregulation of tenogenic markers Scleraxis (Scx) and Tenomodulin (Tnmd) with cyclic tensile stain, crowding and hypoxia appeared to downregulate Tnmd, while crowding upregulated Scx after 7 days. Additionally, H&E staining revealed that cell density decreased from 3 to 7 days of culture for fibers subjected to macromolecular crowding or hypoxia, though no change in cell density was observed with their combined stimulation. Col-1 and Col-3 immunostaining, and fractal analyses, were used to assess matrix content and alignment of hypoxic fibers, compared to normoxic controls, and showed that Col-3 increased by 3 days, while Col-1 content decreased, and Col-1 alignment increased after 7 days. These findings suggest that hypoxia may improve engineered tendon biomechanical properties by promoting early fibrillogenesis, and improved collagen alignment through matrix remodeling. Together, this work provides novel insight into applications of macromolecular crowding and hypoxia to enhance biomechanical maturation of engineered tendon, which could also inform other tendon engineering approaches.