Biomimetic cell containing scaffold for bone tissue engineering

Objectives:To reduce the bone regeneration period in bone tissue engineering, it is desirable to create a more biomimetic microenvironment, which enhances bone formation in the material. The use of a biomimetically mineralized, 3D cell scaffold might be a means of achieving this aim. We enhanced biomineralization in bone marrow stromal cell (BMSC) culture in 3D fibrin gel, and evaluated its material characteristics. Methods: Fibrin gel (j=6mm, L=10mm) was fabricated by mixing a fibrinogen solution containing 2x104 mouse BMSCs with thrombin solution. This composite gel was cultured in an osteogenic differentiation medium in a floating condition. A fixed and stained thin-section of the composite gel was used to evaluate the distribution of matrix protein and minerals. The crystal growth of minerals in the gel was evaluated from SEM images. Results: Von Kossa staining indicated that mineralization in the gel started on day 35. Image analysis of the cross-section indicated that the mineralized area increased from 0% on day 28 to 58.9±12.2% on day 42. The distribution of mineralized area in the cross-section correlated with matrix protein distribution, and the area gradually spread out from each individual cell. The size of the total gel became smaller depending on the culture period, and the contraction rate was affected by the number of cells in the gel. SEM images indicated that the minerals in the gel had a spheroidal morphology with aggregation of needle-like crystals, and that the spheroid grew during the culture period. Conclusion: An in vitro mineralization system in fibrin gel was established using BMSCs. The mineral content and mineral distribution in the gel were dependent on cell number and culture period. This system may be useful in understanding the mineralization process and the development of a new cell/organic/inorganic composite scaffold for bone tissue engineering.