Studies on the formation mechanism and the structure of the anisotropic collagen gel prepared by dialysis-induced anisotropic gelation.

We have found that dialysis of 5 mg/mL collagen solution into the phosphate solution with a pH of 7.1 and an ionic strength of 151 mM [corrected] at 25 °C results in a collagen gel with a birefringence and tubular pores aligned parallel to the growth direction of the gel. The time course of averaged diameter of tubular pores during the anisotropic gelation was expressed by a power law with an exponent of 1/3, suggesting that the formation of tubular pores is attributed to a spinodal decomposition-like phase separation. Small angle light scattering patterns and high resolution confocal laser scanning microscope images of the anisotropic collagen gel suggested that the collagen fibrils are aligned perpendicular to the growth direction of the gel. The positional dependence of the order parameter of the collagen fibrils showed that the anisotropic collagen gel has an orientation gradient.

Multiscale analysis of changes in an anisotropic collagen gel structure by culturing osteoblasts.

Mimicking the complicated anisotropic structures of a native tissue is extremely important in tissue engineering. In a previous study, we developed an anisotropic collagen gel scaffold (ACGS) having a hierarchical structure and a properties gradient. In this study, our objective was to see how cells remodel the scaffolds through the cells-ACGS interaction. For this purpose, we cultured osteoblastic cells on ACGS, which we regarded as a model system for the cells-extracellular matrix (cell-ECM) interaction. Changes in the ACGS-cell composites structure by cell-ECM interactions was investigated from a macroscopic level to a microscopic level. Osteoblastic cells were also cultured on an isotropic collagen gel (ICGS) as a control. During the cultivation, mechanical stimuli were applied to collagen-cell composites for adequate matrix remodeling. Confocal laser scanning microscope (CLSM) was used to observe macroscopic changes in the ACGS-cell composite structure by osteoblastic cells. Small-angle X-ray scattering (SAXS) measurements were performed to characterize microscopic structural changes in the composites. Macroscopic observations using CLSM revealed that osteoblastic cells remained only in the diluted phase in ACGS and they collected collagen fibrils or formed a toroidal structure, depending on the depth from the ACGS surface in the tubular diluted phase. The cells were uniformly distributed in ICGS. SAXS analysis suggests that collagen fibrils were remodeled by osteoblastic cells, and this remodeling process would be affected by the structure difference between ACGS and ICGS. These results suggest that we directly regulate cell-ECM interaction by the unique anisotropic and hierarchical structure of ACGS. The cell-gel composite presented in this study would promise an efficient scaffold material in tissue engineering.