Marsit, Nagi Mohammed Abdalla
(2019)
Characterisation of vacuum dried amniotic membrane and validation of the processing protocol.
PhD thesis, University of Nottingham.
Abstract
Amniotic membrane (AM) has been used for several decades as a wound healing material in different clinical applications. AM is prepared for usage in contact with damaged tissue, therefore, sterility is a crucial issue in AM manufacturing. There is no consensus on the optimal method for preparing and sterilising AM. AM obtained aseptically from elective caesarean section should have low bioburden, thus, a powerful processing and sterilisation methods is potentially not required. Therefore, development of a gentle processing protocol for AM can overcome the limitations of other processing methods such as cryopreservation and radiation sterilisation on AM structure and function.
Vacuum dried AM (VDAM) is a dry AM material manufactured by a novel low temperature drying method, developed previously by Academic Ophthalmology, University of Nottingham. It is not exposed to freezing, nor is it terminally sterilised by irradiation. Instead, a combined cocktail of antibiotics is used during processing to confirm VDAM sterility and the AM is dried at room temperature. It can then be rehydrated prior to or during contact with the body, for example by the tear film when used in ophthalmology. Thus, VDAM incorporates unique qualities over dried AM products presented in literature and currently on the market. Herein, the effects of this unique manufacturing process on VDAM microstructure, biophysical properties and ability to remove high bacterial loads are explored and validated alongside investigation into VDAM stability and antibacterial activity over long term storage conditions.
This project investigated first the macrostructural alterations using histological staining, then microstructural changes using immunohistochemistry and scanning electron microscopy. VDAM thickness, transparency and hydration behaviour were assessed and compared to fresh and cryopreserved AM. The results showed that the VDAM histological and structural properties were maintained. VDAM thickness, transparency, rehydration efficiency and solution permeability of VDAM were efficient.
The VDAM processing protocol was validated for the removal of a deliberately applied high bacterial contamination and any potential antibacterial efficacy investigated. It was shown that each of the sequential VDAM processing steps significantly contributed to lower the contamination level throughout the processing. The vacuum dehydration and antibiotic sterilisation steps were efficiently removed a high load of staphylococcus epidermidis applied ahead of VDAM essential processing steps.
A study on the long-term storage effect on structural and antimicrobial activity of VDAM were also carried out showing that VDAM structural components such as collagen and GAGs and antibacterial potency were maintained during long storage time of over two years.
This research provides a novel conclusion that low temperature vacuum dehydration and antibiotic decontamination can be considered as sterilisation method for production of dried AM, without compromising its microstructure or biophysical characteristics. Furthermore, VDAM is proven microbiologically stable in cold storage for more than two years duration. Therefore, owing to these qualities VDAM can be recommended as biological treatment for ocular surface disorders.
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