• Chikezie Ihesiulor

Student thesis: Master of Philosophy


Abstract Background Atomic Force Microscopy (AFM) imaging and biomechanics on bulk ex vivo human scleral tissue demonstrates the effect of chemical cross-linking on the nanomorphology of collagen fibrils, quantified using 2D-Fast-Fourier Transform. This study sets out to understand scleral role in myopia development and treatment by revealing the effect of cross-linking on scleral structure and mechanics. Methodology Four post-mortem longitudinally oriented scleral strips 7mmx3mm of healthy human scleras (2 male and 2 female donors; average age of 54.5 years) were used. They were randomly assigned into treatment groups (equatorial and posterior regions) each cross-linked with 0.5mM genipin and 0.1mM glutaraldehyde. Both cross-linkers mixed with Phosphate Buffered Saline Gibco™ Dulbeccos Phosphate-Buffered Saline (DPBS) with calcium, magnesium for specific number of hours at room temperature. The control groups (from same scleras and regions) were untreated. After dissection, samples were washed with sterile distilled water, dried and stored in the fridge (4oC) overnight to stick onto a sterilized slide for AFM. The donors were free from diabetes mellitus/ HIV/MRSA. Scleras were obtained from the Manchester Eye biobank (NRES ethics 15/NW/0932). All the scleras had research permission from the donors’ relatives where transplantation was not done. One-way ANOVA between and within the means of the treatment and control groups was done to determine the F-statistics and P-value. Histograms, bar graphs and box and whisker plots were used to show correlations in the different groups. 2-dimensional Fast-Fourier Transform was carried out on the images to calculate the D-periodicities of the tissue collagen fibrils. Results Although some AFM investigations were not completed, significant increase in biomechanical stiffness (higher reduced-YM) by up to 1.3-, 1.2- and 5.2-folds (F1 = 27.64, p = 2.0323E-11; F2 = 80.91, p = 1.6161E-28; F3 = 213.26, p = 1.0364E-46) was observed with genipin crosslinking in three samples. Whereas a 2-fold increase in stiffness occurred in one of the glutaraldehyde cross-linked samples (F3 = 213.26, p = 1.0364E-46). Also, a 2-fold increase in biomechanical stiffness was recorded after 12 hours of incubation with genipin (F1,4 = 36.88, p = 0.00000001). No significant change was found in the D-periodicities (F1,2 = 3.70, p = 0.19). However, the fibril ‘packing’ changed more significantly with genipin than with glutaraldehyde cross-linked samples. Conclusion Chemical cross-linking with genipin significantly alters collagen nanomorphology and biomechanics of the sclera, therefore presenting as a potential treatment intervention for human myopia. More samples should be studied, and the concentration/time of treatment considered in future work.
Date of Award1 Aug 2022
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorMichael Sherratt (Supervisor) & Hema Radhakrishnan (Supervisor)

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