Freezing-induced Microstructural Changes Of Collagen Scaffolds

Understanding the effects of freezing on the functional properties of tissues is critical to cryopreservation of native and artificial tissues, and prognosis after cryotherapies. Although investigations have been performed to correlate macroscopic properties such as mechanical properties with freezing parameters, the effects are typically tissue-type dependent and no physical understanding is available to explain these effects. Since the mechanical and optical properties of a tissue are based on the characteristics of its extracellular matrix (ECM), understanding and quantifying these changes could assist in predicting the response a tissue will have to freezing under different circumstances. In this study, the microstructural changes induced to a type I collagen tissue equivalent construct after freezing to different end temperatures were visualized using macroscopic observation, as well as optical microscopy and scanning electron microscopy. The changes were then quantified using image analysis. The study found that freezing results in a thinner and transparent gel at the macroscopic level. Optical microscopy of the frozen region portrayed apparent "wrinkles" in the matrix, and scanning electron microscopy illustrated a sparser, more disorganized scaffold of fibers, though the fibers appeared to be thicker. Image analysis results indicate that freezing causes a significant increase in matrix porosity and fiber diameter.