In Situ Characterization of the Structural Changes Induced By Acidity Fluctuations in Hydrated Collagen Hydrogels

Collagen is the main component of the extracellular matrix (ECM). In the ECM, The collagen forms in vivo a complex network and a hydrated gel-like environment, which can be mimicked in vitro using a collagen hydrogel. Water plays a crucial role in the self-assembly of collagen fibers by forming hydrogen bonds between residues of the polypeptide chain. The collagen structure can be affected by many factors and is directly related to its function. As a significant part of the ECM, collagen may experience changes in the chemical environment in pathological conditions. For example, in proximity to solid tumors, the ECM acidity increases, while in chronic wounds, it decreases. To mimic ECM initially under healthy physiological conditions that then changed to pathological, we characterize structural changes that collagen hydrogel undergoes when its pH changes from neutral to acidic or basic while maintaining physiological temperature and ionic strength. We investigate the hydrogel structure as closely as possible to its native hydrated state to avoid possible conformational changes during drying. Using Fourier Transform InfraRed Spectroscopy (FTIR) without drying, we show that the collagen secondary structure does not change when the pH changes from neutral to acidic or basic. Cryo Scanning Electron Microscopy (Cryo-SEM) of high-pressure frozen and freeze-fractured collagen gel reveals its submicrometer structure. When the neutral collagen pH decreased, its fibrils were shorter and lacked spatial orientation. The fibrils were helical, longer, and spatially oriented when the pH increased. When returning from acidic to neutral collagen pH, the original structure of the collagen fibers was restored. These findings may contribute to a deeper understanding of the mechanistic basis of pathological conditions in which the collagen structure plays a role in disease progression. In addition, insights from this study may help optimize collagen-based scaffolds for specific tissue engineering applications.