Versatile Wedge-Based System for the Construction of Unidirectional Collagen Scaffolds by Directional Freezing: Practical and Theoretical Considerations

Aligned unidirectional collagen scaffolds may aid regeneration of those tissues where alignment of cells and extracellular matrix is essential, as for instance in cartilage, nerve bundles, and skeletal muscle. Pores can be introduced by ice crystal formation followed by freeze-drying, the pore architecture reflecting the ice crystal morphology. In this study we developed a wedge-based system allowing the production of a wide range of collagen scaffolds with unidirectional pores by directional freezing. Insoluble type I collagen suspensions were frozen using a custom-made wedge system, facilitating the formation of a horizontal as well as a vertical temperature gradient and providing a controlled solidification area for ice dendrites. The system permitted the growth of aligned unidirectional ice crystals over a large distance (>2.5 cm), an insulator prolonging the freezing process and facilitating the construction of crack-free scaffolds. Unidirectional collagen scaffolds with tunable pore sizes and pore morphologies were constructed by varying freezing rates and suspension media. The versatility of the system was indicated by the construction of unidirectional scaffolds from albumin, poly­(vinyl alcohol) (a synthetic polymer), and collagen-polymer blends producing hybrid scaffolds. Macroscopic observations, temperature measurements, and scanning electron microscopy indicated that directed horizontal ice dendrite formation, vertical ice crystal nucleation, and evolutionary selection were the basis of the aligned unidirectional ice crystal growth and, hence, the aligned unidirectional pore structure. In conclusion, a simple, highly adjustable freezing system has been developed allowing the construction of large (hybrid) bioscaffolds with tunable unidirectional pore architecture.