| Summary: | 碩士 === 中國文化大學 === 生物科技研究所 === 101 === Corneal inflammation and injury can result in scarring and loss of transparency of the cornea, which accounts for million cases of blindness worldwide. Corneal transplant surgery is used to remove a diseased cornea and replace it with a clear donor cornea but sometimes this ends up in corneal injury by high-speed particles, which may penetrate your eye and injure deeper structures and causes wounds. Therefore here used a different approach and scrutinized a hydrogel comprised of Hyaluronic acid and Pluronic F-127, which is able to transition between liquid (below 150C) and semi solid (370C) and can be injectable through 25 gauge needle for treating corneal wounds. An in situ formed cross-linked hydrogel is more hydrophilic, insistent and stipulating better biocompatibility. The physico-chemical properties such as transparency, visco-elastic properties, biodegradation & swelling properties, Fourier transforms infrared spectroscopy (FTIR) and scanning electron microscopy was conducted for investigating the best preparation of hydrogel. Results demonstrated that the H1:F15 & H1:F20 hydrogel exhibited thermo responsive rheological properties, biodegradation & swelling properties, which implies H1:F20 hydrogel incubating under the lysozyme solution was still remained 60% of resistant for 7 days. Moreover, here the purpose of this study is to do comparison between effect of these two ratios of H1:F15 and H1:F20 (Hyaluronic acid (H): Pluronic F-127 (F)) of hydrogel on bovine corneal endothelial cells (BCECs) repair with and without porcine platelet rich plasma (P-PRP) and the potential of this copolymer for corneal endothelium reconstruction will be demonstrated by encapsulating BCECs. Numerous cell studies like cytotoxicity, invasion, cell death, cell cycle and cellular junctions performed for mimicking BCECs with hyaluronic acid and Pluronic F-127 copolymer, which proves that it is best scaffold for corneal wounds repair. MTT assay for in vitro cytotoxicity of hydrogel on BCECs and its results shows that ratio H1:F15 with P-PRP showing high cell viability, the results of migration assay through cell culture insert also demonstrate that cell migration is high in H1:F15 with P-PRP. Flow cytometry used to investigate apoptosis and cell cycle study and immunofluorescence for expressing tight junction and gap junction, which also contributed positive effect on BCECs with hydrogel supplemented with P-PRP. Hence these results validate that the copolymer is both non-toxic and biodegradable which proves that BCECs transplanted by the hydrogel can repair, survive and retain normal morphology. Furthermore, these results provide an opportunity for corneal endothelium reconstruction based on tissue engineering approach by the hyaluronic acid and Pluronic F-127 copolymer.
In summary, this tissue engineering centered research for reconstructing corneal endothelium through in situ formed copolymer providing better vision worldwide.
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