[Intellectual contribution]

Development of collagen vitrigel useful for tissue regeneration

Toshiaki TAKEZAWA

Transgenic Animal Research Center

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   The fibril density of traditional collagen gel (Fig. 1) is quite low in comparison to that of connective tissue in vivo. Therefore, we aimed to develop a novel collagen scaffold with high fibril density. In addition, it was known that opaque egg white could be converted into a thin, transparent, rigid glass-like material by boiling and evaporating the moisture — a phenomenon known as vitrification of denatured proteins. We applied this vitrification technology to a traditional collagen gel (Fig. 1). Collagen vitrigels with high fibril density were successfully prepared by a three-step process: a gelation step in which a type-I collagen sol forms an opaque and soft gel by incubation at 37C; a vitrification step in which the gel becomes a rigid material after sufficient drying; and a rehydration step with supplemental moisture that converts the vitrified material into a thin and transparent gel membrane with enhanced gel strength. In this report, we define a vitrigel as a gel in a stable state produced by rehydration following vitrification of a traditional hydrogel. The collagen vitrigel obtained possesses excellent protein permeability (Fig. 1).
   Furthermore, we also prepared frameworkembedded collagen vitrigel scaffolds by inserting a nylon membrane ring into the collagen sol prior to the gelation step (Fig. 2). Anchorage-dependent cells can be cultured on both surfaces of the scaffold by the manipulation of two-dimensional cultures, resulting in the reconstruction of a three-dimensional organoid (Fig. 2). In studies of a crosstalk model between PC-12 pheochromocytoma cells and L929 fibroblasts, we found that nerve growth factor secreted from L929 cells passed through the collagen vitrigel scaffold and induced the neurite outgrowth of PC-12 cells by its paracrine effect (Fig. 3). We also found that a collagen vitrigel scaffold containing vascular endothelial growth factor (VEGF) showed a sustained release of VEGF in vitro and that its subcutaneous transplantation into a rat resulted in remarkable angiogenesis (Fig. 4). These data suggest that the collagen vitrigel scaffold is useful for paracrine assays in vitro and drug delivery systems in vivo. Therefore, we expect that collagen vitrigels could contribute to studies in regenerative medicine and drug development and could serve as an alternative to animal experiments.

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Fig. 1  Preparation methods of a traditional collagen gel and a novel collagen vitrigel and comparison of their properties

Fig. 2  Advantages of the culture technology utilizing a support material-embedded collagen vitrigel membrane scaffold

Fig. 3  Paracrine effect between different types of cells via a collagen vitrigel membrane scaffold

Fig. 4  Drug delivery system utilizing a collagen vitrigel membrane scaffold

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