| Objective:1. To construct recombinant human type-Ⅲcollagen (RHC-Ⅲ)/poly [(3-(methacryloylamino) propyl) dimethyl (3-sulfopropyl) ammonium hydroxide] (PMP DSAH) interpenetrating polymer network (IPN) (RHC-III/PMPDSAH IPN) hydrogel in vitro, and to test the biological and mechanical properties of the material.2. To evaluate the biocompatibility of RHC-III/PMPDSAH IPN hydrogel in rabbit cornea and the feasibility and safety as a cornea substitute.3. To evaluate the effect of never growth factor (NGF) on corneal wound healing after the lamellar keratoplasty in rabbit eye and the feasibility of method of adding NGF into RHC-Ⅲ/PMPDSAH IPN hydrogel.Methods:1. RHC-Ⅲ(10%) were mixed with (3-(methacryloylamino) propyl) dimethyl (3-sulfoproyl) ammonium hydroxide (MPDSAH) at 0℃, then cross-linking agent was added. The mixed solution was then put into the mold, and series of steps were performed, and then, the shaped material was preserved to later use at 4℃.2. The ready RHC-III/PMPDSAH IPN was lyophilized, and then was immersed into the 500μl NGF-bovine serum albumin (BSA) solution whose concentration was 30μg/ml at 4℃for 3-4 days. Then the material was washed with phosphate buffer solution (PBS) and lyophilized, then preserved to later use at 4℃3. According to different corneal materials, one hundred and five rabbits were randomly divided into 3 groups:RHC-Ⅲ/PMPDSAH IPN group (group A,30 rabbits), RHC-Ⅲ/PMPDSAH IPN with nerve growth factor (NGF) group (group B, 30 rabbits), and allograft group (group C,30 rabbits), the donor corneas of group C were offered by the rest 15 rabbits (30 eyes). According to different time points, each group was divided into the group 3rd day, group 1st week, group 2nd week, group 1st month, group 3rd month and group 6th month, with 5 rabbits in each group, the right eyes were operated. Three kinds of materials were implanted into the anterior lamellar of rabbit corneas. Slit lamp was used to observe whether there was severe inflammation after the operation, and transparency of the cornea, corneal neovascularization (NV) and the time of corneal epithelialization were recorded. Hematoxylin-eosin (HE) staining were taken at the 3rd day,1st week,2nd week,1st month,3rd month and 6th month after the surgery, the cornea luminousness were tested and immunohistochemistry means were taken 6 months later. Statistic method was used to compare these data,.P<0.05 was considered that there was a significant difference.4. Twenty-four rabbits were randomly divided into group A and B, with 12 rabbits in each group. According to different time points, each group was divided into the group 2nd week, group 1st month, group 3rd month and group 6th month, with 3 rabbits in each group, the right eyes were operated. The RHC-Ⅲ/PMPDSAH IPN with 5ng/mg NGF and RHC-Ⅲ/PMPDSAH IPN without NGF were implanted into the anterior lamella of the rabbit corneas in each group. Corneas were selected at all the time points after the surgery,3 corneas of contralateral eyes in group A at 6th month were selected as the normal control. Photochemical gold chloride impregnations were performed to observe the regeneration of corneal nerves under light microscope at all the time points. The number of the nerve fibers in corneal epithelium and anterior lamellar layer were calculated. Statistic method was used to compare the number in two groups, and P<0.05 was considered that there was a significant difference.Results:1. The material was 12mm in diameter,200μm thick, and the curvature radius was 7.25 millimetres, equilibrium moisture content of the hydrogel was (90.2±0.7)%, luminousness was (89±1)%, refractive index was 1.35, tensile strength (0.56±0.41) MPa, the properties of RHC-III/PMPDSAH IPN with NGF was similar to the RHC-III/PMPDSAH IPN, drug loading was 5ng/mg, and entrapment efficiency was 10.2%.2. The grafts had been all retained during the experiment without severe rejection. There was no significant differences in the transparency of the cornea, NV and cornea luminousness among the 3 groups at the 6th month (F=0.07, F=0.07, F=1.48, P>0.05). The group B achieved reepithelialization first in 5.00±0.59 days, and there was a significant difference among the 3 groups (F=57.99, P<0.05). HE staining results demonstrated that implanted materials could integrate into the host tissue well and support corneal epithelialization. Collagen fibrils arranged regularly 6 months later. Immunohistochemistry results showed the positive expression of AE5 which is the antibody of keratin-3 which is a marker protein of the corneal epithelial cells.3. The nerve staining showed there were already nerve regeneration in the corneal epithelium and anterior lamellar layer in two groups at the 2nd week, at the 1st month, some regenerated nerve fibers in the superficial stroma formed a network, at the 3rd month, the number of regenerated nerve fibers in the two groups increased obviously, more networks and lateral branches were formed,6 months later, the number of regenerated nerve fibers in group A were more than group B, and the structures were more normal in group A. Some nerve fibers did not regenerate in both groups, and the number of regenerated nerve fibers in two groups were far less than the normal control. There were significant differences in the number of the nerve fibers between group A and B at all the time points (t=13.57,t=15.71, t=38.22, t=34.11,P<0.05)Conclusion:1. The transparency and refractive index of the material were good, although the mechanical strength was a little poorer, which was need to enhance.2. Although there was a good biocompatibility of it in rabbit cornea with high safety, RHC-III/PMPD SAH IPN could not replace the allograft completely because of the little poorer mechanical strength.3. NGF could promote the corneal wound healing after the lamellar keratoplasty.4.The method of adding NGF into the material to form a robust compound tissue-engineered corneal was very feasible and effective, and show some creativity, which has not been seen in other paper in our country.5. The photochemical gold chloride impregnation is a good histological examina-tion of corneal nerve instead of confocal microscopy. |
PDF Full Text Request