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Title: Development of a synthetic substrate for conjunctival cell transplantation
Author: Makuloluwa, Aruni Kumari
ISNI:       0000 0004 6495 9480
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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The conjunctiva is a translucent mucous membrane consisting of epithelial cells and goblet cells (GCs). It protects the eye from external insults and prevents desiccation of the ocular surface by producing mucins of the tear film. Severe scarring due to injuries and diseases could lead to its anatomical and functional impairment. Moreover, the success of a limbal or a corneal transplant is dependent on the presence of a healthy ocular surface and a tear film. Various grafts and substrates employed to replace the damaged conjunctiva to date, have advantages and disadvantages peculiar to the material. Therefore, there is scope for the development of a better substrate for conjunctival cell transplantation. Understanding the protein composition of the extracellular matrix (ECM) of the conjunctiva that regulates cellular functions, could guide the development of a substrate. The aims of the present study, therefore, were to determine the role of ECM proteins on the growth of conjunctival cells and to investigate cell growth and behaviour on surface modified poly-epsilon-lysine (PεK) hydrogels. The ECM proteins of a conjunctival epithelial cell line, HCjE-Gi cell line, were isolated using 1% ammonium hydroxide. The protein composition of the cell-secreted ECM from days 1 to 42 was determined using immunofluorescence and mass spectrometry experiments. The growth of cells on cell-secreted ECM of days 3 to 28 over seven days was determined by immunocytochemistry (ICC). The maximum adsorption of type IV collagen, laminin 111 and fibronectin onto tissue culture polystyrene (TCP) was determined using indirect enzymelinked immunosorbent assays. The adhesion and growth of cells over seven days on TCP with pre-adsorbed type IV collagen, laminin 111, fibronectin and α-2-Heremans Schmidglycoprotein (α-2-HS-GP) were determined by ICC. The expression of stem cell (SC) and GC protein markers over fourteen days on TCP with pre-adsorbed proteins were determined by ICC. The cell adhesion, growth and expression of MUC5AC protein over fourteen days on PεK hydrogels with and without pre-adsorbed proteins were determined by ICC staining. Type XVIII collagen, a structural collagen, was present in the ECM of HCjE-Gi cells in vitro. The peptides of chains that give rise to laminin 332 were the most abundant glycoproteins present in the ECM. The peptide abundance of α-2-HS-GP was highest in the day 1 ECM, whereas that of fibronectin, nephronectin and vitronectin were highest in the day 7 ECM. Perlecan and agrin were the most abundant proteoglycans in the ECM. The day 7 ECM contained the highest proportion of growth factors and their binding proteins. The proportion of proteases and their inhibitors increased and decreased, respectively, with time. The adsorption of type IV collagen and fibronectin onto TCP reached a saturation point when pre-adsorbed from 5μg/mL solutions, whereas the adsorption of laminin 111 onto TCP reached a saturation point when pre-adsorbed from a 0.5μg/mL solution. Type IV collagen and fibronectin increased the adhesion, and type IV collagen, α-2-HS-GP and cellsecreted ECM of days 3 and 5 increased the growth of HCjE-Gi cells. The expression of SC and GC protein markers reduced and increased, respectively, in vitro. Azelaic and suberic acid cross-linked PεK hydrogels supported the adhesion and growth of HCjE-Gi cells ex vivo. However, pre-adsorption of proteins onto PεK hydrogels did not affect the adhesion or the growth compared to those without pre-adsorbed proteins. There was evidence of GC differentiation on azelaic acid cross-linked PεK hydrogels with and without pre-adsorbed proteins and on suberic acid cross-linked PεK hydrogels with pre-adsorbed fibronectin. PεK hydrogels are a promising substrate for the ex vivo expansion of conjunctival cells for conjunctival cell transplantation purposes. Functional peptides of ECM proteins identified to enhance the growth and differentiation of conjunctival cells may be employed to develop a substrate for conjunctival cell transplantation in the future.
Supervisor: Williams, R. L. ; Kaye, S. B. ; Stewart, R. M. K. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral