Exogenous mineralization of cell-seeded and unseeded collagen-chitosan hydrogels using modified culture medium

Induced biomineralization of materials has been employed as a strategy to increase integration with host tissue, and more recently as a method to control cell function in tissue engineering. However, mineralization is typically performed in the absence of cells, since hypertonic solutions that lack the nutrients and culture components required for the maintenance of cell viability are often used. In the present study, we exposed fibroblast-seeded three-dimensional collagen-chitosan hydrogels to a defined culture medium modified to have specific concentrations of ions involved in biomineralization. The modified medium caused a significant increase in calcium deposition in collagen-chitosan gels, relative to constructs incubated in a standard medium, though serum supplementation attenuated mineral deposition. Collagen-chitosan constructs became opaque over 3 days of mineralization in modified Dulbecco's modified Eagle medium (DMEM), in contrast to translucent control gels incubated in standard DMEM. Histological staining confirmed increased levels of mineral in the treated constructs. Rheological characterization showed that both the storage and loss moduli increased significantly in mineralized materials. Mineralization of fibroblast-seeded constructs resulted in decreased cell viability and proliferation rate over 3 days of incubation in modified medium, but the cell population remained over 75% viable and regained its proliferative potential after rescue in standard culture medium. The ability to mineralize protein matrices in the presence of cells could be useful in creating mechanically stable tissue constructs, as well as to study the effects of the tissue microenvironment on cell function.