Effects of Hydrodynamic Ozonated Water Processing on the Thermal Stability and Structural Integrity of the Human Amniotic Membrane

This study aimed to verify the morphology, biochemical composition, and thermal characterization of hydrated human amniotic membrane (HAM) processed in an ozonated water hydrodynamic system. This is an in vitro experimental study in which HAM samples were divided into two groups: in natura (IN) and ozonated (O3). Analyses were performed using histology, Fourier-transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA/DTG), and differential scanning calorimetry (DSC/dDSC). Ozonation for 40 min preserved the biochemical integrity of HAM, maintaining the characteristic vibrational bands of Amides I, II, and III. Histological analysis showed morphological changes in epithelial cells, with partial removal in some regions, while the basement membrane and the scaffold remained preserved. Thermal analysis revealed that the in natura sample presented a bimodal dehydration profile, with a first event occurring between 60 and 65 °C associated with the evaporation of free or weakly bound water, and a second event peaking around 80 °C related to the removal of structural water. In contrast, the ozonated HAM exhibited a unimodal profile, with the mass loss peak shifted to approximately 70 °C. These findings were corroborated by DSC analysis, which showed a reduction in denaturation temperature from approximately 85 °C in the in natura sample to around 75 °C in the ozonated sample. The dDSC analysis confirmed the transition from a bimodal to a unimodal behavior after treatment, indicating a reduced energy barrier for protein denaturation and lower thermal stability of the collagen matrix. These results suggest that ozonation promotes alterations in the epithelial layer, which may favor the loss of both free and bound water. It is concluded that processing with ozonated water induces structural modifications, especially in the epithelial layer, and reduces the thermal stability of hydrated HAM without significantly altering the biochemical signature of collagen. This approach shows potential as an alternative method for membrane processing; however, functional evaluations are required to confirm its clinical applicability.