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

Abstract

The efficiency of amniotic membrane (AM) transplantation in different types of ocular surface disorders is due to its outstanding properties such as antifibrotic, antibacterial, anti-inflammatory and antiangiogenic, working as a versatile scaffold to promote corneal tissue epithelialization. A proper preparation, preservation and clinical application are crucial for the best outcomes in the treatment of different severe ocular disorders, taking into account its fragility. In this context, by combining high-sensitivity tools such as atomic force microscopy (AFM) and Fourier transform infrared (FTIR) spectroscopy with histological and immunohistochemical examination, we aimed to investigate the ultrastructural modifications of the amniotic membrane (AM) upon UV exposure and/or antibiotic treatment, with relevance for clinical applications in ocular surface surgery. From the morphological point of view, we noticed a loss of cuboidal cells in the basal membrane, accompanied by the splitting of collagen fibers upon UV and/or gentamicin treatment, while structural alteration of proteins was evidenced by the FTIR quantitative analysis of the secondary structure. A decrease in α-helix and β-sheet content, accompanied by increased content in less ordered structures (turns, random and side chains), was noticed after all the treatments. At the nano-scale, AFM details showed modifications of collagen fibrils in terms of their thickness and network compaction upon gentamicin and/or UV treatment. The enzymatic digestion assay demonstrated that UV exposure significantly reduces the degradation rate of the AM, while gentamicin treatment promotes an accelerated enzymatic digestion upon UV exposure. In order to highlight the clinical impact of the research, a clinical case is presented showing the relevance of amniotic membrane transplantation in pterygium surgery.

The Collagen Origin Influences the Degradation Kinetics of Guided Bone Regeneration Membranes

“…These findings are consistent with previously described alterations that were found after water degradation of human collagen [ 26 ]. Previous studies correlating the membrane structure with degradation reported that thicker collagen fibers, higher collagen density and a more compact structure may be produce slower biodegradation [ 27 , 28 ].…”

“…Additionally, the impact of embedded cells on membrane degradation is missing. Despite the limitations of the study, our in vitro findings are intended to give a first estimation of the membranes behavior, but it should also be considered that in vivo, a more complex set of different cell types, body fluids and possible microbial contamination may influence the membrane degradation process, with the oral cavity environment being an scenario that is difficult to mimic [ 27 , 28 ]. Therefore, it might be challenging to directly transfer these data to the clinical practice.…”

In Vivo Biological Evaluation of Biodegradable Nanofibrous Membranes Incorporated with Antibiofilm Compounds

“…The drawbacks based on collagen include insufficient mechanical properties and unpredictable degradation profile that may compromise the in vivo performance [ 51 ], furthermore, present risk of disease transmission [ 7 ]. Although different treatments have been reported to enhance biomechanical properties and stability of collagen matrices through natural crosslinking, the addition of plasma rich in growth factors and UV exposure [ 52 , 53 , 54 ], ethical and religious factors associated with high costs had directed to studies of alternative materials. Polymeric membranes, however, are biodegradable, biocompatible, and exhibit, in general, superior degradation rates.…”

Collagen-Based Biomaterials in Periodontal Regeneration: Current Applications and Future Perspectives of Plant-Based Collagen

“…From the literature, natural biopolymers with a porous micro-scale are more suitable for angiogenesis, nutrient transport, and mineral deposit for bone formation [ 45 , 46 ]. In contrast, nano-scale membranes are excellent for cell attachment and differentiation, and being inhibitory barriers against physical damages and pathogens to maintain the microenvironmental niche for bone regeneration [ 47 , 48 ]. Different ultrastructures also affect their mechanical and chemical properties; a more compact and denser ultrastructure shows a higher time for chemical degradation as indicated by trypsin degradation of a dense structure of multi-directional networks in a Jason ® membrane compared to a network of collagen fiber bundles of a Biocollagen ® membrane.…”