The complex supramolecular architecture of collagen biopolymer plays an important role in tissue development and integrity. Developing methods to report on collagen structures assembled in vitro would accelerate the pace of utilizing it in biomedical applications. Employing imaging techniques and turbidity measurements, we mapped the light scattering properties of 3D collagen hydrogels formed at initial concentrations of 1 mg ml?1 to about 5 mg ml?1 and several incubation temperatures. The transmission electron microscopy (TEM) images show that collagen scattering features consist of both native-like fibrils and filamentous structures that do not have the characteristic fibrillar striation observed in this protein. Spindle-shaped fibrils appear at the concentrations of 1, 2, 2.5 and 4 mg ml?1 and the spiral-shaped fibrils are formed at the concentrations of 2 and 2.5 mg ml?1. The multiphoton microscopy (MPM) images reveal that in the 3D collagen hydrogels a unified relationship between second harmonic generation (SHG) signal directionality and fibril morphology and/or sizes is not likely. The MPM images, however, showed important micro-structural details. These details lead us to conclude that the dependence of SHG signals on the number of interfaces created upon assembly of 3D collagen hydrogels can account for the strength of the detected backscattered signals...
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