| ObjectiveTo study the release characteristics and effect of a new medical biomaterial—Drug delivery-fibrin glue-amniotic membrane. To approach its feasibility and superiority using in ocular surface graft and tissue repair, provide experimental foundation for the clinical application in the future. The study includes three parts:Experiment I: Study on the release characteristics of bFGF-fibrin glue-amniotic membrane (bFAM) in vitro.Experiment II: Study on the effect of bFGF-fibrin glue-amniotic membrane for corneal alkali burn.Experiment III: Study on the release characteristics of ganciclovir -fibrin glue-amniotic membrane (GFAM).MethodsExperiment I: We use fibrin glue compounded with bFGF and amniotic membrane to prepare the bFAM (1ng/ml). ELISA method was used to measure the content of bFGF. And In vivo conditions were simulated to study the release characteristics of bFAM. Experiment II: We use fibrin glue compounded with bFGF and amniotic membrane to prepare the bFAM (2.5μg/ml). 45 New Zealand rabbits were used to make corneal alkali burn models and divided into 3 groups: group A: bFAM group, group B: amniotic membrane group (AM), group C: Alkali burn and untreated group(AG), each group have 15 rabbits. The round paper was soaked in 1mol/L NaOH, then put it on the surface of rabbit cornea. 4~6 hours after alkali burn, we did bFAM grafting or amniotic membrane grafting. After operation, we observe the linical curative effect. corneas have the clinical examination on slit-lamp, observing the detachment of amniotic membrane, corneal edema and epithelial wound healing, scoring for the corneal neovascularization, transparency and corneal fluorescein staining. 7d,14d and 28d after operation, 5 corneas were extracted for histopathological analysis. The expression of bFGF in corneas was measured by immunohistochemistry analysis.Experiment III: In vitro study, we use fibrin glue compounded with GCV and amniotic membrane to prepare the GFAM(5mg/ml) and study its release characteristics. In vivo study, GFAM transplantation was performed on 25 eyes of the rabbits as experimental group and 1mg/ml GCV eye drops 100ul was given in 5 eyes as the control. After that the aqueous humors of experimental group were collected at 1d, 2d, 3d, 4d and 5d, the aqueous humors of comparison group were collected at 1.5 hours after the fifth eye drop, then assayed by high performance liquid chromatography (HPLC).ResultsExperiment I: In vitro release study revealed that the accumulative release rate of bFGF was (41.85±4.45)% 24 h after incubation and it rose to (61.5±4.49)% 48 h later,The release process went to a balance condition 96 h later.Experiment II:1.Detachment of amniotic membrane from cornea: In the day 5 after operation, amniotic membrane dissolved gradually, some of them dropped from cornea. In the day 14 after operation, much of the bFAM dissolved and dropped.2.The score of coreneal transparency: Compared with group C, the score of corneal transparency in both group A and group B in the day 28 after alkali burn showed significant difference(P<0.05).The score of corneal transparency in the day 28 showed significant difference between group A and group B(P<0.05).3.The score of CNV: Compared with group C, the score of CNV in both group A and group B in the day 28 after alkali burn showed significant difference(P<0.05). But in the day 14, the score of CNV did not have significant difference between each group(P>0.05).4.The healing of corenal epithelial: In both group A and group B, the epithelial healing is faster than group C in the day 14 and 28 after alkali burn(P<0.01), and the epithelial healing showed significant difference between group A and group B(P<0.05).5.The expression of bFGF: The expression of bFGF in group A is much higher than group B and group C in the day 7 afer operation (P<0.01), and in other time points the expression have not distinct difference in all groups(P>0.05).Experiment III:1.In vitro release study revealed that the accumulative release rate of GCV was (45.67±5.32)% 24 h after incubation and it rose to (63.42±4.68) % 48 h later, The release process went to a balance condition 96 h later.2.In vivo study indicated that the drug level in aqueous of comparison group is (0.501±0.040)μg/ml. at 1d, the drug level in aqueous of experimental group is (0.613±0.037)μg/ml, the drug level in aqueous is significantly higher for experimental group than comparison group(P<0.05); at 2d, the drug level in aqueous of experimental group is (0.554±0.093)μg/ml, it is higher than comparison group, but the difference showed no statistics meaning(P>0.05); at 3d, the drug level in aqueous of experimental group is (0.443±0.043)μg/ml, it is lower than comparison group, the difference showed no statistics meaning(P>0.05); the GCV level in aqueous of experimental group become lower than that of comparison group at 4d and 5d , the difference have statistics meaning (P<0.05).ConclusionOur study is in the initial stage, including the release characteristics of bFAM and GFAM in vitro, effect of bFAM for corneal alkali burn, the conclusion as follow:1. bFAM takes on a sustained release property in vitro.2. Compared with amniotic membrane, bFAM shows better curative effect for corneal alkali burn. The bFAM can maintain in the surface of corneal wound for a long time, accelerate the healing of epithelial, play an important roll in prohibitting cornel dissolution and perforation, suppressing the formation of neovascularization. It also can reduce scar and improve corneal transparency.3. GFAM takes on a sustained release property both in vitro and in vivo, it can achieve an effective drug concentration and provide experimental foundation for the treatment of viral keratitis in the future.
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