Lazutina, Elena
(2017)
Investigating potential biochemical properties of fetal membrane spongy layer for clinical application at the ocular surface.
PhD thesis, University of Nottingham.
Abstract
The amniotic membrane, well known scaffolding tissue, which widely uses and benefits of having anti-inflammatory, anti-microbial, anti-fibrosis, anti-scarring with low immunogenicity and reasonable mechanical properties.
Amniotic Membrane Transplantation (AMT) is an established treatment modality, which favourably influence ocular surface re-epithelisation and prevents angiogenesis, thus promoting healing and minimising scarring. It is also used at several other sites of the body. Despite its widespread use, key elements of the membrane and its precise mechanism(s) of action remain to be elucidated. Unfortunately, over the years conflicting clinical reports have suggested variations in the efficacy of AM utility. Conventional methods for amnion preparation do not acknowledge the presence of the SL.
My project is a continuation of previous PhD completed in the department, which mentioned the Spongy Layer as one of the important layers in Amniotic Membrane, which had not been look for in any previous work and Dr A Hopkinson, the author of previously mentioned PhD, accidentally discovered possibility to separate that layer from the amnion, it had been decided to take a close look at the layer and investigate properties.
Researchers at the University of Nottingham have developed and improved techniques of manufacturing clinical grade the amnion and they have identified the SL as a substance that is variably present in conventional amnion. They have developed techniques to entirely isolate the SL, which allows comprehensive characterisation of its composition and biological properties.
The project originally designed to investigate all layers of Amniotic Membrane separately in comparison with amniotic membrane which is completely free from Spongy Layer (SL detached) as well as Spongy Layer attached to the amniotic membrane (classically used layer) and identify a layer which is richest in proteins and growth factors. So, three different samples were investigated:
1) Isolated Spongy Layer;
2) Isolated Amniotic Membrane;
3) Amniotic Membrane with Spongy Layer attached (classical layer, which well- known used).
I used technique developed in the department. This technique allows separating the SL without unnecessary mechanical tissue disturbance and isolated SL was used to investigate comprehensive characterisation of the composition and biological properties.
Our technique of removing SL is simple and could be easily adapted. The SL imbibes water well and significantly expands, which makes it thick and easy to pull using forceps or using blunt edge of the scalpel blade to push the SL from the amnion surface without mechanical interruption.
Investigation of origin of the Spongy Layer during gestation period done through intensive literature search and came to conclusion that the Spongy Layer developed from the extraembryonic endoderm. It is well known that the SL acts as a barrier between vascular amnion and avascular chorion.
Also, question was about similarities and differences of embryological origin of the Wharton Jelly and the Spongy Layer. The present in which of TGFb1 (immunofluorescent staining) could be result of cross link during the embryological development.
As previously, reported by Dr A Hopkinson et al in 2006, that the Spongy Layer’s biochemical composition is containing TGF-b1, EGF and HGF. The structure of SL is reported to be composed of Collagen types I-VIII, the SL contains high level of hyaluronan, which is a major carbohydrate component of the ECM.
To extract proteins from the SL, a few different techniques were used, first of all the tissue weighted, freeze dried and lyophilised in buffers, which were different and depended on the experiment planned.
After, proteins were analysed through the Searchlight protein array analysis, 2-D protein quantitation, the Bradford (Commassie staining) assay, the mass spectrometry, had been discovered that the SL contains angiogenic factors, biomarkers, cell adhesions factors, cytokine proteins, growth factors, metalloproteinases, chemokine proteins, neurotrophic factors and cellular components. Experiments show that the level of those factors and proteins in fresh AM, the Transplant Ready Amniotic Membrane (TRAM) and the SL shows that significant amount of proteins was simply washed out during preparation from the TRAM, however the SL is holding those proteins in significant level probably due to imbibing while absorbing water.
The important discovery of this project was the cytotoxic effect of the SL and its antimicrobial properties.
Some fractions of the SL with high molecular weight proteins show apoptotic activity to corneal keratofibroblasts by necrosis rather than apoptosis. However, cells occurred apoptosis after treatment with lower molecular weight proteins. As preliminary data shows the SL to be cytotoxic, this could lead to some understanding of different outcome in the Amniotic Membrane transplantation (AMT). However, this area needs further investigation.
Well known antimicrobial properties of the amniotic membrane were established only in the TRAM and in the samples of the SL which were prepared in the same way as TRAM (washed in gentamicin), however samples of the SL which were not washed in gentamicin did not show antimicrobial properties.
One of the next steps of investigation properties of Spongy Layer was the measurement of the thickness of the layer in normal physiological situation, during gestation. As the SL is imbibing the water quickly, in vitro it was difficult to measure “real” thickness of the membrane; this gave an idea to measure it in vivo. To answer the question of “real” (physiological) thickness of the Spongy Layer, different methods were used, however the Ultrasound technique was our method of interest, as it gave the possibility to see the layer during gestation without any interruption and see changes in thickness prior to delivery. These measurements were done in the Fetal Maternal Medicine Department by very experienced sonographer. The Spongy Layer has a variable thickness in three different areas (cervical part, mid region and apical part of the uterus) the SL regions had been divided accordingly.
The Spongy Layer has a variable thickness depending on the anatomical location. The difference of the SL thickness had been measured in vivo (ultrasound technic – described below) and in vitro (this work published by JJ Gicquel) the compared results matched.
The major problem of the project was the separation of proteins and to break hyaluronic chains to extract clean proteins.
In my project, I used two different techniques to separate proteins according the molecular weight of proteins presented in the sample.
1) Revers Phase Solid Phase Extraction (RP-SPE) isolation of proteins; and
2) Soluble Protein Fractionation using Vivaspin columns
The second technique in my hands was more successful and I decided to use this for all my samples. To minimise the possibility of sample variations from the point of sample preparation of the SL and the AM itself, I used the same technique for all experiments and combined samples.
Nevertheless, my samples were not 100% clear due to different protein structure and shape.
This layer therefore has the potential to be exploited clinically for the treatment of several indications. However, before it can be employed, the layer requires further investigation to determine characterise the content of potential factors. In addition, as the spongy layer is predominantly composed of mucin and proteoglycans resulting in a gelatinous/viscous substance, a processing procedure must be developed to either modify the substance in a usable format, or to extract the beneficial factors, for clinical use.
Being able to demonstrate the isolated SL and its derivatives can be exploited as potential therapeutic agents in the treatment of many ocular surface disorders, would have significant translational potential. The control of inflammation caused by disease (e.g. Ocular cicatricial pemphigoid) and any injury (e.g. Chemical burns) and the limitation of scarring and vascularisation would preserve sight or allow successful secondary intervention such as corneal drafting, which otherwise has a high risk of failure in such situations, the potential for preventing visual impairment and promoting quality of life in all age groups in therefore immense.
My work proved that the SL is a separate layer and is having a vast number of different factors in a significantly high amount compare to amnion itself.
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