Quantifying the ultrastructure changes of air-dried and irradiated human amniotic membrane using atomic force microscopy: a preliminary study

Abstract

Air-dried and sterilized amnion has been widely used as a dressing to treat burn and partial thickness wounds. Sterilisation at the standard dose of 25 kGy was reported to cause changes in the morphological structure as observed under the scanning electron microscope. This study aimed to quantify the changes in the ultrastructure of the air-dried amnion after gamma-irradiated at several doses by using atomic force microscope. Human placentae were retrieved from mothers who had undergone cesarean elective surgery. Amnion separated from chorion was processed and air-dried for 16 h. It was cut into 10 × 10 mm, individually packed and exposed to gamma irradiation at 5, 15, 25 and 35 kGy. Changes in the ultrastructural images of the amnion were quantified in term of diameter of the epithelial cells, size of the intercellular gap and membrane surface roughness. The longest diameter of the amnion cells reduced significantly after radiation (p < 0.01) however the effect was not dose dependent. No significant changes in the shortest diameter after radiation, except at 35 kGy which decreased significantly when compared to 5 kGy (p < 0.01). The size of the irradiated air-dried amnion cells reduced in the same direction without affecting the gross ultrastructure. At 15 kGy the intercellular gap decreased significantly (p < 0.01) with Ra and Rq, values reflecting surface roughness, were significantly the highest (p < 0.01). Changes in the ultrastructure quantified by using atomic force microscope could complement results from other microscopic techniques.

Nano-Scale Modifications of Amniotic Membrane Induced by UV and Antibiotic Treatment: Histological, AFM and FTIR Spectroscopy Evidence

“…An increase in fibril thickness was noticed upon gentamicin treatment, depending on the concentration: 100 width/16 nm height for AG40, and 110 width/15 nm height for AG80, the length of collagen chains being well preserved within the 3D network. Notable changes were identified when UV exposure was applied immediately after gentamicin treatment (AGUV), in terms of collagen chain fragmentation, accompanied by increased thickness of collagen fibrils (180 width/29 nm height), although the D-band pattern for short collagen fragments was not altered, which is in concordance with previously reported data [39,45,46]. However, we could not find in the literature any reference related to the antibiotic influence on ultrastructural changes of the amniotic membrane, except our previous work [18], in which we demonstrated that gentamicin treatment is more favorable compared to ciprofloxacin, by assessing the denaturation process using FTIR spectroscopy.…”

“…The authors showed that after crosslinking treatment, the AM underwent biomechanical and ultrastructural changes: the brittleness was increased, the hardness was enhanced and the bamboo-like fiber morphology was changed. Another study was performed in order to examine ultrastructural images of the surface morphology of the air-dried and gamma-sterilized AM observed under AFM and to quantify possible changes in epithelial cell size, intercellular gap size and surface roughness [46], but with no relation to the collagen matrix. In this case, AFM results confirmed that both gamma radiation and air drying caused a reduction in cell sizes (about 32.3% reduction in the average longest diameter), but the changes did not affect the gross morphology of the membrane.…”