De-Epithelization of the Human Amniotic Membrane Using a System Involving Ozonated Water and Ultrasound

Abstract

The aim of this study was to evaluate whether a system involving ozonated water and ultrasound causes de-epithelization of the human amniotic membrane (HAM). The experiment protocol was carried out in four stages. Stage I was carried out to determine the duration of the experiment. Stage II comprised the first experiment, involving four groups of samples studied in triplicate: control/natural (IN), processed with ultrasound in a liquid medium (US), processed with ozonated water (O3), and processed with ozonated water combined with ultrasound (US_O3). Stage III was performed to confirm the results, following the same steps present in Stage II. Stage IV involved the use of oxygen to confirm the hypothesis. Histological analysis was carried out to verify whether the effects of O₂ were similar to those of O₃. The system was activated, and ozonation was carried out for 10 min, as in the previous experiment, reaching a concentration level of 3.0 mg/L. The samples were submerged and positioned in the reservoir and processed separately for 55 min. The biochemical properties were assessed using Fourier transform infrared spectroscopy, and the morphology was examined using histology and scanning electron microscopy. The spectra of the samples exhibited similarities; however, subtle changes were highlighted, such as smooth band shifts and intensity changes. The morphology indicated that ultrasound achieved more efficient HAM de-epithelialization compared to ultrasound combined with ozonated water and ozonated water alone. One plausible hypothesis for this observation is that cavitation represents the primary mechanism responsible for de-epithelialization. When ultrasound is combined with ozone, the bubbles generated by ozone gas reduce the cavitation effect. This study is pioneering as it demonstrates an ultrasound system capable of the efficient de-epithelialization of the HAM.

Keywords: amnion; biomaterials; de-epithelization; human amniotic membrane; ozone; tissue engineering; ultrasound.

Figure 1

Experiment protocol steps.

Figure 2

O₃ concentration curve dissolved in water at 18 °C.

Figure 3

FT-IR spectra obtained from the analyzed samples: IN—Control/natural (black). US—Processed with ultrasound in liquid medium (red). O3—Processed with ozonated water (green). US_O3—Processed with ozonated water combined with ultrasound (blue).

Figure 4

Histological images of the samples: (A) IN—Control/natural. (B) US—Processed with ultrasound in a liquid medium. (C) O3—Processed with ozonated water. (D) US_O3—Processed with ozonated water combined with ultrasound. The arrows (1.a), (2.b), (1.c), and (1.d) indicate epithelial cells. The arrows (2.a), (3.b), (2.c), and (2.d) indicate the basement membrane. The arrows (4.d), (3.c), and (3.d) indicate areas with epithelial cell removal. The arrows (3.a), (1.b), (4.c), and (4.d) indicate the stroma.

Figure 5

SEM-FEG images of the samples: (A) IN—Control/natural. (B) US—Processed with ultrasound in a liquid medium. (C) O3—Processed with ozonated water. (D) US_O—Processed with ozonated water combined with ultrasound. Arrows (1.a), (2.c), and (1.d) show intact epithelial cells. Arrows (2.b), (3.b), and (3.c) show irregular epithelial cells. Arrows (1.b), (1.c), and (2.d) show regions with epithelial cell removal.

Figure 6

Histological images of the samples: (A) IN—Control/natural. (B) O2_US—Processed with water, oxygen, and ultrasound. (C) US—Processed with ultrasound in a liquid medium. Arrows (1.a), (1.b), and (3.c) indicate epithelial cells. Arrows (2.a), (3.b), and (4.c) indicate the basement membrane. Arrows (2.b), (1.c), and (2.c) indicate areas with epithelial cell removal.