Cells sense and respond to the mechanical properties of the substrate to which they adhere. Matrices composed of collagen have been an important experimental system to study cell responses to the stiffness of the extracellular matrix. We had previously reported the development of thin films composed of collagen fibrils which are several hundred nanometers thick and offer excellent optical and handling characteristics. Vascular smooth muscle cells respond to the thin films very similarly to bulk gels of polymerized collagen in their spreading and proliferation. Dehydrating the fibrils increases their contact stiffness, and correspondingly cells were shown to spread more and proliferate on the dehydrated thin films. We now present detailed Atomic Force Microscopy analysis of the effect of dehydration on these thin films. In situ dehydration on the atomic force microscope (AFM) stage and in a tightly controlled environment over a 24h period, allowed us to minimize thermal drift and observe changes in the topography and stiffness in the same regions of the thin film due to dehydration. The thin films are comprised of large fibrils on top of a bed of, underlying bed of small fibrils. There was a change in the size of collagen fibril diameter due to the loss of water. We could independently determine the Elasitc modulus of the two layers using a beam on an elastic foundation model for large fibrils, and calculate the effective stiffness of the entire thin film. The calculated stiffness was found to agree well with measured effective stiffness of the entire thin film using a colloidal probe modified AFM tip. Our findings detail the effects of dehydration on the collagen thin film system, and offer insights into the role of hydration in affecting the topographic and mechanical properties of polymerized collagen in general.
Pub Type: Journals
Atomic Force Microscopy, Collagen Type I, Dehydration, Mechanical properties