| Background:In glaucoma surgery, post-operative fibrous tissue hayperplasia and scar adhesions leads to bleb scarring, collagen sediment, formation of scars, then decreasing bleb function, which is the major cause of failure for glaucoma surgery. Based on the pathophysiological characteristics of bleb scarring after trabeculectomy, numerous specialists at home and abroad adopted many methods to reduce the formation of bleb scarring. In recent years, owing to the combination of new technology, complications have been remarkably decreased and the success rate for surgery raised. The surgery known as compound trabeculectomy includes: improvement of operative modes and technique, and introduction of non-penetrating trabeculectomy; external placement in surgery of removable sclera sutures, and post-operative controlling quantitative removal or laser release of sclera sutures and so on; drugs used in or after surgery for inhibition of scarring formation; application of implant in surgery.Non-penetrating trabeculectomy resects the corneal stroma of sclera subsurface as deep sclera, outer wall of Schlemm tube, anterior trabecula and Dieldrin anterior, retaining natural trabecula Dieldrin membrane as filtration layer, implanting hyaluronic acid in sclera layers, making aqueous humor meet some resistance in its smooth extravasation, gradually lowering intraocular pressure, and maintaining the integrity of the eye; thus forming a space within the sclera, making aqueous humor flow and drain via different outflow channels. The advantages of the surgery are that operative complications are fewer; vision recovers more quickly; post-operative inflammation is mild; filtering blebs are more diffuse and flatter; the incidence of eye within the eye is lower.Currently, only5-Fu and MMC are maturely used in clinic for prevention of scarring prevention. As one of the earliest antimetabolites used in clinic, Mitomycin C has strong anti-proliferative effect, which is100times that of the same dose of5-Fu, remarkably inhibiting the proliferation of fibroblasts, slowing down the process of wound healing, improving the success rate for eye surgery with poor prognosis. Its effect has been verified by clinic and evidence-based medicine.Chitosan is a fine, degradable natural bio-material, having good histocompatibility and functions of stopping bleeding and inhibiting bacteria, being capable of adjusting immune functions, promoting tissue repair, inhibiting the proliferation of tissue, reducting scar adhesions and so on. It belongs to a category of important controlled release drug delivery system, extensively used in clinic for prevention of post-operative adhesions with bone joints and abdominal cavity, and used also as ophthalmic viscoelastic.Based on the above study and review of literature, we hope to integrate antifibrotic agents in the implant, that is, develop chimeric mitomycin C chitosan sustained-release sponge, carrying out in vivo and in vitro animal studies so as to effectively inhibit proliferation of fibroblasts, reduce formation of bleb scarring and remarkably raise the success rate for glaucoma surgery.Chapter I Preparation of chimeric mitomycin C chitosan sustained-release sponge Objective:By combining drugs and materials by means of pharmaceutics, we prepared stable chimeric mitomycin chitosan sustained-release sponge and doubled its anti-scar adhesion effect, preventing post-operative adhesions of filtration road scar in glaucoma from three dimensions and solving the problem of operative failure after surgery in glaucoma.Methods:(1) Choose chitosan with high degree of deacetylation as basic raw material and compound medical chitosan via freeze dehydration, then preparing chitosan sponge;(2) Integrate the two materials by adopting secondary freeze-drying technology, making the product porosity larger and the aperture smaller, more conducive for drugs to fill and load.Statistical treatment:By means of SPSS13software, one-way ANOVA LSD, SNK were adopted, and sponge wet and dry states and its thickness were analyzed, which revealed that there was no remarkable difference in the figures between the groups.Outcome:(1) Successfully prepared new-type different-concentration chimeric mitomycin C loaded slow-release sponge.(2) Successfully detected the in vitro drug release of the sponge.(3) Achieved fine biodegradability and biocompatibility for the sponge.Conclusion:(1) Successfully prepared new-type different-concentration chimeric mitomycin C loaded slow-release sponge.(2) Chimeric mitomycin C loaded slow-release sponge achieved fine biodegradability and biocompatibility, which provided a theoretical basis for it to be used as implanted material.Chapter â…¡ Experimental study on mitomycin slow-release sponge promoting fibroblast apoptosisObjective:To ascertain the bioactivity for mitomycin in mitomycin slow-release sponge we prepared, verify that its controlled release system is able to promote fibroblast apoptosis and explore the lowest concentration for mitomycin effectively to inhibit the proliferation of fibroblasts, so as to provide a experimental basis for further animal experiment and to reduce complications.Methods:(1) Adopting tissue explant adherent culture and culturing primary human Tenon’s capsule fibroblasts.(2) Collecting stable4to6generations of human Tenon’s capsule fibroblasts, using CCK-8assay to detect the growth-inhibiting function of human Tenon’s capsule fibroblasts. Observing and photographing under microscope the cell morphology and numbers.(3) Detecting cell apoptosis by PI staining and flow cytometry.Statistical treatment: By means of SPSS13software, data were analyzed by ANOVA. Analysis of variance pairwise comparisons were done by LSD. P value<0.05indicated that the difference was statistically significant.Outcome:(1) Successfully cultured primary human Tenon’s capule fibroblasts and confirmed it by immunofluorescence.(2) The sample containing MMC had remarkable inhibiting effect on human Tenon’s capsule fibroblasts. As the concentration of MMC increased in the sample, the inhibition rate for cell growth gradually increased. And the inhibition rate with the concentration50μg/ml of MMC-SPCM group corresponded to that with100μg/ml of MMCgroup.(3) MMC affected the morphology of human Tenon’s capsule fibroblasts, changing the normal cell shape, reducing the number of cells and increasing apoptosis and necrosis of cells. (4) It was found by cell cycle analysis via flow cytometry that as the concentration of mitomycin was higher, the apoptosis rate for human Tenon’s capsule fibroblasts was remarkably increased, indicating that mitomycin slow-release sponge group(MMC-SPCM group) promoted the apoptosis of fibroblasts through its lower concentration.Conclusion:(1) Human Tenon’s capsule fibroblasts were successfully cultured in this part of the experiment.(2) The activity of mitomycin in the mitomycin sponge prepared by us proved to be normal, which was capable of promoting the apoptosis of fibroblasts and inhibiting the proliferation of fibroblasts.(3) By means of CCK-8assay and flow cytometry, the impact of different concentrations of mitomycin C was inspected and compared on apoptosis rate, indicating that lower concentration (125μg/m) of mitomycin chitosan slow-release sponge still had inhibiting effect on the proliferation of human Tenon’s capsule fibroblasts, which showed its slow-release effect.(4) This laid a basis for mitomycin chitosan slow-release sponge to be applied in the next in vivo animal experimental study, for post-operative adhesions in anti-glaucoma surgery.Chapter III Mitomycin slow-release sponge for prevention of post-operative scarring in glaucoma-Its in vivo experimental studyObjective:To investigate the effect of Mitomycin slow-release sponge in in vivo experimental study on post-operative scarring in glaucoma.Methods:(1) A group of30healthy New Zealand rabbits were randomly allocated into groups of NS, SPCM and MMC-SPCM, all of which underwent non-penetrating trabeculectomy on one eye. After that, under sclera flap was injected saline respectively and placed SPCM sponge sheet and MMC-SPCM sponge sheet.(2) Post-operatively at Id,3d,5d,1w,2w,4w,8w,12w were observed the general condition, the height and area of filtering bleb and changes in intraocular pressure.(3) Post-operatively at10d and28d,2rabbits in each group were sacrificed and18at3m, HE staining was done of them respectively and observation made of pathological changes in them.(4) Post-operatively at10d,2rabbits in each group were sacrificed and real time PCR was done. And mRNA of VEGF and TGF-β2were detected in the3groups.Outcome:(1) With NS group, SPCM group and MMC-SPCM group, post-operatively under slit-lamp were observed the general conditions of conjunctiva, cornea, anteroom and other anterior segments, in which there was no remarkable difference noted.(2) After surgery with time extension, the size and height of the filtering blebs gradually became smaller. Between the3groups was done ANOVA with pair wise comparison technique, and post-operatively at14d, lm,3m, there was remarkable difference(P<0.05) in the height and area of filtering blebs.(3) In comparison between NS group, SPCM group, and MMC-SPCM group, the post-operative intraocular pressure showed remarkable difference(P<0.05) in the operation group of both eyes and non-operation group(control group); ANOVA was done between the3groups with pair wise comparison. Post-operatively at14d, lm,3m (Figure3-7), there was remarkable difference(P<0.05)in the changes of the intraocular pressure.(4)By HE staining and immunohistochemical staining of the filtration road areas of the groups, which were then observed at10d and28d. At the time of10d, in and under conjunctiva, and in filtration road area were noted large amounts of inflammatory cell (mainly neutrophilic granulocytes) infiltration, with marked congestion expansion in blood vessels and also seen new vessels and fibroblasts. Inflammatory cells in filtration road area were obviously decreased than before; collagen were more closely arranged; and fibroblasts were increased than previously. Post-operatively at28d, NS group was found with large amounts of collagenous fibre proliferation, densely arranged, with filtration road basically congested.(5) PCR results revealed that mRNA of VEGFå'ŒTGF-β2in MMC group was by far lower than those in PBS group and slow-release membrane group. The difference between them was statistically significant (P<0.001). However, in comparison between slow-release membrane group and PBS group, the difference in mRNA of VEGF or TGF-β2was not statistically significant, indicating that chitosan sponge containing MMC could markedly inhibit the mRNA content of VEGF and TGF-(32.Statistical treatmentExperimental data were expressed with mean±tandard deviation (mean±SD). SPSS13.0software was used for statistical analysis. Comparison between averages of the two samples was done with t test; comparison between averages of multiple samples were done with ANOVA; pair wise comparison between multiple samples was done with LSD or S-N-K. Each experiment was repeated at least3times, P＜0.05denoting remarkable significant difference.Conclusion(1) In this part of experiment, we successfully created New Zealand rabbit non-penetrating trabeculectomy model and in accordance with experiment content the experimental animals were allocated into groups.(2) Observation of general condition of anterior segments in post-operative animals indicated that topical application of mitomycin slow-release sponge had no remarkable effect on ocular tissues, showing its good histocompatibility.(3) After application, mitomycin slow-release sponge, saline group and pure chitosan sponge group showed remarkable difference in the changes of post-operative intraocular pressure and filtering blebs, indicating that application of mitomycin slow-release sponge could prevent post-operative filtration road adhesions in glaucoma.(4) Pathohistological examination revealed that topical application of mitomycin slow-release sponge could reduce scar formation by reducing inflammatory reaction in local tissue.(5) PCR results showed that MMC slow-release sponge inhibited scar adhesions by inhibiting mRNA of VEGF and TGF-β2at the operative site, which proved the effectiveness of topical application of mitomycin polyactic acid controlling membrane. |
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