Microrheological Characterization of Collagen Systems: From Molecular Solutions to Fibrillar Gels

Abstract

Collagen is the most abundant protein in the extracellular matrix (ECM), where its structural organization conveys mechanical information to cells. Using optical-tweezers-based microrheology, we investigated mechanical properties both of collagen molecules at a range of concentrations in acidic solution where fibrils cannot form and of gels of collagen fibrils formed at neutral pH, as well as the development of microscale mechanical heterogeneity during the self-assembly process. The frequency scaling of the complex shear modulus even at frequencies of ∼10 kHz was not able to resolve the flexibility of collagen molecules in acidic solution. In these solutions, molecular interactions cause significant transient elasticity, as we observed for 5 mg/ml solutions at frequencies above ∼200 Hz. We found the viscoelasticity of solutions of collagen molecules to be spatially homogeneous, in sharp contrast to the heterogeneity of self-assembled fibrillar collagen systems, whose elasticity varied by more than an order of magnitude and in power-law behavior at different locations within the sample. By probing changes in the complex shear modulus over 100-minute timescales as collagen self-assembled into fibrils, we conclude that microscale heterogeneity appears during early phases of fibrillar growth and continues to develop further during this growth phase. Experiments in which growing fibrils dislodge microspheres from an optical trap suggest that fibril growth is a force-generating process. These data contribute to understanding how heterogeneities develop during self-assembly, which in turn can help synthesis of new materials for cellular engineering.

Spatially resolved microrheology of heterogeneous biopolymer hydrogels using covalently bound microspheres

“…The ensembleaveraged elastic moduli, determined using the average elastic moduli from 1 to 4.1 rad/s, scaled with a power-law of 1.7 ± 0.3, which is comparable to previous findings ranging from 1.7 to 2.2 (Yang et al 2009;Motte and Kaufman 2013). The magnitudes of the elastic modulus obtained using NHS-microsphere are comparable to previous results ranging from 0.1 Pa at 1 mg/ml to 50 Pa at 5 mg/ml (Velegol and Lanni 2001;Latinovic et al 2010;Shayegan and Forde 2013). The slight variation in power-law scaling and elastic modulus magnitude with previous findings can be due to the difference in manufacturer, type, and batch of collagen used.…”

“…From the observations of previous sections, NHS-microsphere is potentially a viable option to probe the local environment of the heterogeneous 3D gels. To verify that the NHS-microsphere is sensitive to changing microenvironment, experiments were carried out to track approximately 40 microspheres in collagen gel, a natural biopolymer that has been found to show high degree of heterogeneity (Velegol and Lanni 2001;Parekh and Velegol 2007;Latinovic et al 2010;Shayegan and Forde 2013), with varying concentrations. As shown in Fig.…”

Intact Telopeptides Enhance Interactions between Collagens

“…1 b, and a detailed description can be found in (22). An optical tweezers instrument was used for microrheology assays (22,24,25).…”

“…1 b, and a detailed description can be found in (22). An optical tweezers instrument was used for microrheology assays (22,24,25). Thermally induced shortrange position fluctuations of optically trapped probe particles (polystyrene microspheres) were detected using a quadrant photodiode at a bandwidth of 100 kHz.…”

“…2) (26); for this reason, the values of G 0 (f) presented in this work extend only up to 4 kHz. Furthermore, in our system the elastic modulus (G 0 ) contains information on elasticity of both the trap and the medium, and was corrected by removing the trap's contribution (determined from the lowest-frequency measured value of G 0 independently for each bead; (22,26). This correction method is supported by the apparent low-frequency plateau in G …”