AbstractIt is now well accepted that the mechanical properties and cell adhesion profile of 2D and 3D extracellular matrix molecules combine to dictate cellular fate processes, such as differentiation, migration, proliferation, and apoptosis, through a process generally known as 'mechanotransduction', or the conversion of mechanical signals into a cellular response. The stiffness and adhesion density combine to affect the force balance that exists between an adherent cell and the surrounding substrate. We have established BioMEMS, microfluidic technology to alter the mechanical properties and cell adhesion profile of collagen scaffolds. Using soft lithography, we fabricate elastomeric networks that serve as conduits for the controlled mixing of type I collagen solutions. Our technology enables us to generate reproducible, controlled homogeneous and inhomogeneous microenvironments for 3D cell culture, assays of cell behavior in 3D, and the development of bioartificial tissue equivalents for regenerative and reparative therapies. The adhesivity of collagen is modulated by covalently grafting peptides (such as RGD) or proteins (such as albumin) to soluble collagen molecules with 1- ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), a hetero-bifunctional coupling agent. EDC activates the carboxylic group of collagen and forms an amine bond with the grafting molecule. The grafted collagen self-assembles into a fibrillar gel at physiological temperature and pH with no measurable changes in rheological properties compared to controls. A solution of peptide-grafted collagen is then mixed in microfluidic networks with unaltered collagen to form controlled gradients or other patterns of the two solutions, which immobilize upon self-assembly. Separately or in the same network, the mechanical properties of the collagen gel can be altered regionally by the microfluidic delivery a solution of a cell-tolerated crosslinking agent. We use genipin, which has the unique property of generating crosslinks that autofluoresce. The intensity of the fluorescence correlates with the degree of crosslinking (and thus the mechanical properties) enabling us to monitor and measure changes in mechanical properties dynamically and non-invasively. Lastly, though it requires constant delivery or recirculation, the same networks can be used to impose gradients of soluble factors, such as growth factors and cytokines. Thus, we have developed a platform to examine the response of cells to simultaneous chemotactic, haptotactic, and durotactic gradients in a 3D environment. We are employing this technology to examine the response of neural cells to gradients of biomaterial properties to optimize cues for spinal cord regeneration.
Mechanical Properties of Native and Cross-linked Type I Collagen Fibrils