Assessment of pH and temperature effects on collagen gel microstructure using second harmonic generation and scanning electron microscopies

Abstract

Collagen is a fibrous structural protein that possesses a characteristic triple-helical conformation which aids in the stabilisation of molecular confirmations. For example, in tissues collagen aids in maintaining the structural integrity. Collagen accounts for 30% of the total protein content in the body and is a major constituent of the extracellular matrix (ECM). The ECM provides structural support for cellular components and is a key regulator of biochemical functions in the human body related to tissue growth and development. Thus, for the maintenance of normal organ function and to facilitate wound healing, homeostasis of the ECM is crucial. Sustained homeostatic dysregulation results in terminal and pathological conditions such as cancer where structural variation in the ECM is not well understood and is difficult to characterize. In the current research, natural collagen hydrogels synthesized in differing chemical environments were investigated by second harmonic generation (SHG) and scanning electron microscopies (SEM). The gels were synthesized within biologically relevant ranges of polymerization pH and polymerization temperatures to investigate how these variables affect the structure and morphology of collagen fibrils. Results of imaging dried samples with SEM revealed structural parameters of collagen D-banding periodicity of 59.8 ± 4 nm in gels simulating normal physiological conditions and was in line with the ~67 nm periodicity reported for in vivo native-banded fibrils. Qualitative analysis of the hydrogels revealed lower fibril content and thicker fibrils at lower temperatures which was evidenced by higher SHG signals in quantitative analysis. The fibril diameter was maximum at pH 6.5, and lower fibril content was observed at a lower pH. Statistically significant differences were observed in average fibril diameter and in SHG intensities for varied polymerization temperature and pH, demonstrating that the technique can probe collagen microstructure and provide a greater understanding of ultrastructural changes occurring within the ECM.