Time-lapsed nanoscale maps of the elastic modulus of collagen during cross-linking by bimodal AFM

Collagen is the most abundant structural protein in mammals. Collagen in tissues is exposed to cross-linking processes such as glycation which might cause progressive tissue stiffening. Tissue stiffening might be considered a landmark of aging. Yet a quantitative characterization of the elastic modulus of collagen nanofibers under different cross-linking processes and stages is not available. Bimodal AFM was applied to generate time-lapsed maps of Young's modulus of type I collagen nanoribbons under two cross-linking processes associated, respectively, with the presence of ribose and glutaraldehyde in the solution. Elastic modulus maps were acquired for different incubation times (0, 30 min, 12 h, 24 h and 1 week). The experiments were performed in liquid. The Young's modulus showed an initial sharp increase after an incubation time of 30 min, from a few MPa (native) to 100 MPa. From then onwards we measured a monotonic increase until a saturation value of about 2 GPa was reached after one week. We did not observe a dependence on the elastic modulus evolution using ribose <i>versus</i> glutaraldehyde. The saturation value was very similar to that measured on dry collagen nanoribbons.