Development of autologous macrophage-encapsulated gellan fluid gel for the prevention of blindness from ocular surface scarring

Alqahtani, Mohammad Yahya M. (2024). Development of autologous macrophage-encapsulated gellan fluid gel for the prevention of blindness from ocular surface scarring. University of Birmingham. Ph.D.

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Abstract

Background: Corneal scarring is the fourth leading cause of preventable visual loss. Following infection /injury, the cornea undergoes a repair mechanism involving a strong inflammatory response, repair and degradation mechanisms. This results in an alteration of its molecular components and structure, which could potentially lead to scar formation, and ultimately cause blindness. Corneal transplantation is the standard treatment for corneal scarring, which has donors’ shortage and a rejection risk. Thus, the development of a novel treatment is significantly required for maintaining the intact corneal structure. Our group using a novel gellan fluid gel to deliver a single anti-scarring molecule in a sustained manner into the effected cornea showed promising findings. Thus, we proposed using gellan fluid gel to allow sustained release of multiple molecules instead of one to limit inflammation and promote corneal tissue remodelling while minimising fibrosis, which would lead to regenerative healing of the cornea and untimely maintenance of vision. Macrophages are critical players during wound healing repones, meditating infection elimination, damaged cell clearance, resolution of inflammation, and tissue remodelling. Thus, we proposed that administration of a balance of M1 and M2 macrophages at the right time on the ocular surface using gellan fluid gel as an effective delivery system may lead to regenerative corneal wound healing and eventual preservation of sight. The present project aimed to develop autologous macrophage-encapsulated gellan fluid gel for delivering bioactive molecules that aid re-epithelialisation, while minimising fibrotic signalling cascades to aid scarless healing of the ocular surface and the preservation of sight.
Method: Human monocyte derived macrophages with pro-inflammatory (M1), anti-inflammatory (M2), and pro-healing (M2) properties were generated using GM-CSF, LPS+IFN-, and M-CSF, IL-4+IL-13, respectively. The viability of M1/M2 in gellan fluid gel and its effect on macrophage phenotype at 4h were assessed by fluorescent microscopy (FM), and Flow cytometry and qPCR, respectively. To test macrophage polarisation states, M1/M2 were cultured with opposing cytokines, i.e., M1 with IL-4/13, and analysed by RNAseq and Luminex.
Result: The gellan fluid gel maintained both M1/M2 survival and polarisation states. However, the polarisation states of our generated subset of M1/M2 macrophages are not maintained in response to sequential stimulation with positing stimuli.
In conclusion: these results show that gellan fluid gel is biocompatible and can be used to deliver autologous macrophage therapy into the cornea. The instability of the M1/M2 polarisation states necessitated caution when applying them to the cornea with an unstable microenvironment.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):