The effect of chemical and light cross-linking procedures on the levels of beneficial growth factors in human amniotic membrane for use in ophthalmology
Bajwa, Amrita Kaur
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The overall goal of this research is to alleviate suffering and improve visual outcomes for burn patients during recovery, using amniotic membrane as a stabilized 'patch' for the corneal surface. Second and third degree facial burns result in scarring, which can lead to contraction of tissues around the eyelids, a condition called ectropion. This renders the burn patient unable to close their eyes or blink, which can be followed by loss of moisture, breakdown of defense mechanisms, permanent scarring and opacification of the cornea. Instead of continuously applying artificial tears to these victims' eyes, which would actually slow rate of recovery, the use of amniotic membrane would help seal in moisture as well as increase the rate of recovery, while not compromising corneal clarity. However, due to enzymes in the tears of inflamed eyes, the amniotic membrane is degraded fairly rapidly. It is the goal of this team's research efforts to determine an efficient way to stabilize the amniotic membrane by cross-linking its constitutive proteins, so that the rate of amnion degradation by enzymes is decreased but the levels of various beneficial pro-healing factors are preserved. While work is being done to assess the effects of cross-linking on the rate of amnion degradation, this study focused on the effects of protein crosslinking on the levels of transforming growth factor-beta (TGF-ß 1 ), one of the most abundant growth factors in human amniotic membrane. A specific protocol utilizing liquid nitrogen for the homogenization of de-epithelialized and cryopreserved human amniotic membrane (HAM) was created. TGF-ß 1 content in untreated, carbodiimide cross-linked, and photochemical tissue bonding (PTB) cross-linked HAM samples was measured using enzyme-linked immunoasorbent assay (ELISA). It was shown that while the stability of the membrane due to cross-linking of the collagen in the treated samples was increased, the protein content was severely affected; TGF-~1 levels in highly crosslinked HAM samples were extremely lower than that of the untreated. It was also demonstrated via ELISA that decreasing the extent of protein crosslinking by varying the treatment time with carbodiimide or decreasing the fluence of the light used in PTB resulted in equally stabilized amniotic membrane, but higher TGF-ß 1 content than in previously treated samples. This signifies that both chemical and photochemical protocols can result in beneficial collagen cross-linking, and potentially preserve the helpful growth factors already inherent in HAM for use in ophthalmology.
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