Effects of Gelatin Hydrogel Loading Mitomycin C on Conjunctival Scarring in a Canine Filtration Surgery Model

Abstract

Purpose.: To investigate the effects and toxicities of gelatin hydrogel (GH) loading mitomycin C (MMC) on IOP and conjunctival scarring in a canine model of glaucoma surgery in comparison with conventional MMC application.

Methods.: Glaucoma surgery models were made in six beagles. An MMC-loaded GH was implanted under the conjunctiva of one eye (GH-MMC group) and 0.04% MMC-soaked sponges were placed under the conjunctiva of the other eye (MMC group) for 5 minutes. Intraocular pressures and bleb features were then assessed for 4 weeks postoperative, followed by histological evaluation. The ratio of conjunctival area to scleral area, the densities of collagen and the numbers of fibroblasts, vessels, and proliferative cell nuclear antigen (PCNA)-positive cells were then quantified.

Results.: In both groups, IOP reduction and bleb formation were maintained in a similar manner for 4 weeks postoperative. No significant difference in the ratio of conjunctival area to scleral area was found between the two groups. Collagen density and the numbers of fibroblasts and vessels were significantly lower in the MMC-treated group than in the GH-MMC–treated group. No significant difference in PCNA-positive cells was found between the two groups.

Conclusions.: Implantation of MMC-loaded GH ameliorated toxicity to conjunctiva compared with the 5-minute placement of MMC, whereas its effect on IOP reduction and bleb formation was similar. These results suggest that using GH for the application of MMC is a safer method than the conventional application of MMC in glaucoma filtration surgery.

Application of mitomycin C (MMC) after glaucoma surgeries, particularly trabeculectomy, has greatly improved the surgical results via strong suppression of fibroblast proliferation. However, its application also increases the risk of complications, including infectious endophthalmitis,^1–3 as it may produce thin blebs. Therefore, an effective and safer approach to suppressing fibroblast proliferation after glaucoma surgery is currently greatly needed. There have been several studies focused on subconjunctivally implanted drug delivery systems (DDSs) to provide a sustained release of antiproliferative drugs during an extended period after glaucoma surgery.^4–11 The findings of most of those studies have indicated that these DDSs maintained IOP reduction and prolonged bleb persistence in a manner similar to the conventional application of antimetabolites, such as 5-fluorouracil and MMC, while significantly reducing their toxicity. However, those DDSs have yet to produce clinically satisfactory results. 

Gelatin hydrogel (GH), biodegradable material consisting of bioactive proteins, is reportedly a useful DDS in many fields of medicine,¹² as it enables a controlled release of growth factors over a period ranging from 5 days to 3 months.^13,14 To date, this DDS has been applied to numerous clinical therapies.^15–19 We previously reported the possibility of GH application as a new DDS for a longer-term maintenance of filtering blebs without a significant loss of vessels after glaucoma surgery.²⁰ Therefore, the use of GH might offer the ability to apply MMC at a lower concentration, thus inducing minimal damage to physiological structures, including vessels. 

The aim of this present study was to verify, in a canine model of glaucoma surgery, whether MMC-loaded GH produces similar effects on IOP reduction, filtration bleb formation, and cell proliferation and induces less injury near the lesion in comparison with the conventional application of MMC. 

Materials and Methods

Materials and Drugs

The details of the GH preparation was the same as that described in our previous report.²⁰ The freeze-dried GH (MedGel P19) was purchased from MedGEL Co., Ltd. (Kyoto, Japan), and MMC was purchased from Kyowa Hakko Kirin Co., Ltd. (Tokyo, Japan). Preparation of the GH containing MMC was as follows: 0.04% MMC solution was produced by diluting with physiological saline, and a 5 × 5 × 1.5-mm block of the freeze-dried GH was then soaked in the 0.04% MMC solution overnight at 4°C. 

Measurement of MMC Concentration in the GH

The MMC concentration was measured by HPLC.²¹ The GH (treated as described above) was dissolved in 0.5 mL 1N hydrochloric acid for 10 minutes, and then neutralized by 0.5 mL sodium hydroxide. Substances with a molecular weight of less than 5000 were extracted by use of a filter (EMD Millipore, Billerica, MA, USA). Chromatographic analysis was carried out on a 4.6 × 250-mm ID C18 column (Capcell Pak C18 MG S-5; Shiseido Co., Ltd., Tokyo, Japan) connected to an HPLC chromatographic system (LC-10AD; Shimadzu Corporation, Kyoto, Japan) equipped with a UV detector (SPD-10AVP; Shimadzu) set at 365 nm. The mobile phase of acetonitrile:water (1:1, by volume) was delivered at 0.5 mL per minute. We quantified the concentration of MMC dissolving in the GH from a calibration curve that had been obtained beforehand. 

Animals and IOP Measurement

Six beagles weighing 9.0 to 10.0 kg were purchased from Japan SLC (Shizuoka, Japan). Beagles were fed regular dog food, had free access to tap water, and were housed in an air-conditioned room at approximately 23°C and 60% humidity with a 12-hour light-dark cycle. The experimental procedures for the animals were conducted in accordance with the ARVO Statement for Use of Animals in Ophthalmic and Vision Research. The IOP measurements were obtained by use of a calibrated pneumatonometer (Model 30 Classic; Medtronic Solan, Jacksonville, FL, USA) under general anesthesia with intravenous injection of pentobarbital sodium (35 mg/kg body weight) in a front-facing position. 

Glaucoma Filtration Surgery Model

For the glaucoma filtration surgery model, the beagles were anesthetized with pentobarbital sodium as described above. Next, a 10-mm fornix-based flap of conjunctiva and the Tenon's capsule (length, 5 mm) was made as described previously.²⁰ After a 3 × 1-mm scleral portion was removed at the limbus, peripheral iridectomy was performed, followed by closing the conjunctiva with a 10-0 nylon suture. After surgery, the appropriate amount of 3 mg/g ofloxacin ointment was applied to the eye. 

Experimental Protocol

For the experimental protocol, 12 eyes of six beagles were used. In each animal, one eye was treated with GH containing MMC (GH-MMC), while the fellow eye was treated with MMC alone. In the eyes treated with GH-MMC, a 5 × 5 × 1.5-mm block of GH was placed under the conjunctiva during surgery. A sponge (M.Q.A. sponge; Inami Co., Tokyo, Japan) soaked with 0.1 mL 0.04% MMC solution in distilled water was placed under the conjunctiva for 5 minutes in the eyes treated with MMC alone. 

Intraocular pressures, as well as bleb scores, were assessed every 2 weeks for a month, followed by histological evaluation of the eye after the dogs were killed by injecting a lethal dose of pentobarbital sodium. Conjunctival and scleral areas of the lesion were then measured, as described below. Conjunctival collagen density and the numbers of fibroblasts, vessels, and proliferative cell nuclear antigen (PCNA)-positive cells were then quantified. 

Bleb Scores

Blebs were examined via slit-lamp microscopy and graded according to the definition reported by Perkins et al.,²² reflecting bleb height and size as follows: Score 1, minimally high conjunctiva thickening without swelling; Score 2, mild swelling present; Score 3, elevated bleb covering the 2- to 3-o'clock position of the eye; and Score 4, greatly elevated bleb covering more than the 4-o'clock position of the eye. A score of 0 indicated no observable bleb. The blebs were graded in a masked manner by one author (MU) with regard to the treatment given to each eye. 

Histology and Immunohistochemistry

Conjunctival and scleral tissue specimens were fixed with Carnoy Solution (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) and embedded in paraffin. Next, 5-μm-thick sections were cut, mounted on silanized slides (Dako Japan, Kyoto, Japan), and deparaffinized with xylene and a series of graded ethanol. Then, Azan-Mallory staining for each section was used to identify collagen fibers. The conjunctival and scleral areas of the lesion where the flap was made were assessed by use of a computerized morphometry system (MacSCOPE Ver 2.2; Mitani Corporation, Fukui, Japan), and the ratio of conjunctival area to scleral area was then calculated. The collagen density in the conjunctiva was quantified as follows: the collagen area identified by Azan-Mallory staining was quantified by use of a computerized color extraction system (Win Roof Ver. 6.13; Mitani Corporation), and the collagen density was then calculated as the ratio of collagen area to total conjunctival area. 

To retrieve the antigen, the sections were pretreated with 10-mM citrate buffer at pH 6.0, and then autoclaved for 5 minutes at 120°C before immunohistochemical staining. The sections were then soaked in absolute methanol containing 3% hydrogen peroxide for 5 minutes at room temperature to remove endogenous peroxidase activity. Next, the sections were incubated with 10% nonimmune goat serum for 5 minutes to suppress nonspecific binding. For identification of fibroblasts and vessels, anti-bovine vimentin antibody (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and mouse anti-human von Willebrand factor antibody (Wako Pure Chemical Industries, Ltd.), respectively, were used. The sections were incubated overnight at 4°C with each antibody, followed by reaction with appropriate reagents from a streptavidin-biotin peroxidase kit (Dako Denmark A/S, Glostrup, Denmark) and 3-amino-9-ethylcarbazole for 5 to 10 minutes. The sections were then lightly counterstained with hematoxylin. The procedures of immunohistochemical staining with mouse monoclonal antibody against PCNA (PC10) (M0879; Dako Denmark A/S) were as described in our previous report.²⁰ Fibroblasts, vessels, and PCNA-positive cells were counted at the sites where they accumulated in the conjunctival and scleral lesions by use of a light microscope (number per ×100 field), and the average number of each type of cells or vessels in five randomly selected fields was then calculated. 

Statistical Analysis

Each measurement was expressed as the mean ± SD or the mean ± SE. Statistical comparisons for repeated measurements used repeated-measures ANOVA followed by other tests. Bleb scores were statistically analyzed by Wilcoxon's signed ranks test. Other parameters were evaluated by Student's t-test for paired data. Differences were considered statistically significant at a P less than 0.05. 

Results

Concentration of MMC in MMC-Loaded GH

Chromatographic analysis revealed 0.012 mg MMC was included in the 5 × 5 × 1.5-mm block of GH. Because the volume of the MMC solution impregnated in the GH was estimated at 0.0375 mL, the concentration of MMC was deemed to be approximately 0.32 mg/mL. 

Effects of GH-MMC Compared With MMC in the Canine Filtration Surgery Model

The initial values of IOP (mean ± SD, mm Hg) were 20.1 ± 3.7 in the eyes treated with GH-MMC and 20.6 ± 2.3 in the eyes treated with MMC alone. In both the eyes treated with GH-MMC and the eyes treated with MMC alone, IOP was found to be significantly reduced at 2 and 4 weeks postoperative (Fig. 1). The IOP values (mean ± SD, mm Hg) at 4 weeks postoperative were 12.1 ± 4.2 in the eyes treated with GH-MMC and 12.1 ± 5.0 in the eyes treated with MMC alone. There was no significant difference in IOP between the eyes treated with GH-MMC and the eyes treated with MMC alone during the observation period (P = 0.94, repeated-measures ANOVA). 

Figure 1

 

Intraocular pressure changes in the eyes treated with GH-MMC (•) or a 5-minute placement of MMC alone (○). Data are shown as the mean ± SE for six dogs. *P < 0.05; **P < 0.01 versus initial values (paired t-test). n.s.: P ≥ 0.05 (paired t-test). No significant difference was found between the eyes (P = 0.94, repeated-measures ANOVA).

The bleb score remained high at least until 4 weeks postoperative in the eyes treated with GH-MMC as well as in the eyes treated with MMC alone (Table 1). No significant difference in bleb score was found between the eyes treated with GH-MMC and the eyes treated with MMC alone at 2 and 4 weeks postoperatively. 

Table 1

 

Bleb Score Changes in the Beagle Eyes Treated with GH-MMC or a 5-Minute Placement of MMC Alone

 

As shown in Figures 2A and 2B, the conjunctival thickness in the eyes treated with GH-MMC was almost the same as those treated with MMC alone. No significant difference in the ratio of conjunctival area to scleral area was found between the eyes treated with GH-MMC and those treated with MMC alone (Table 2). 

Figure 2

 

Representative photomicrographs of the sections obtained from eyes treated with GH-MMC (A) and MMC alone (B) at 4 weeks postoperative and stained with azan stain. The conjunctiva and the sclera are surrounded by red and blue lines, respectively. Scale bars: 1 mm.

Table 2

 

Comparisons of the Ratio of the Conjunctival Area to the Scleral Area, Collagen Density, and Densities of Fibroblasts, Vessels, and PCNA-Positive Cells Between Eyes Treated With GH-MMC and Eyes Treated With MMC Alone

 

Figures 3A and 3B show the collagen area in conjunctiva with azan staining in the eyes treated with GH-MMC and MMC alone, respectively. The collagen area identified by the color extraction method is shown in green in the eyes treated with GH-MMC (Fig. 3C) and in the eyes treated with MMC alone (Fig. 3D). The paired t-test revealed that collagen density was significantly higher in the eyes treated with GH-MMC compared with those treated with MMC alone (Table 2). 

Figure 3

 

Representative photomicrographs of the conjunctiva obtained from the eyes treated with GH-MMC (A) and MMC alone (B) at 4 weeks postoperative and stained with azan stain. The area in green identified by use of the color extraction method illustrates collagen in the conjunctiva in the eyes treated with GH-MMC (C) and that in the eyes treated with MMC alone (D). Scale bars: 500 μm.

In the eyes treated with GH-MMC, more vimentin-positive cells (i.e., fibroblasts) were found compared with those treated with MMC alone (Figs. 4A, 4B). The densities of fibroblasts in the lesion were significantly higher in the eyes treated with GH-MMC compared with those treated with MMC alone (Table 2). 

Figure 4

 

Representative immunohistochemical staining of section for vimentin in the eyes treated with GH-MMC (A) and those treated with MMC alone (B). Scale bars: 50 μm.

The distribution of vessels in the eyes treated with GH-MMC and in those treated with MMC alone at 4 weeks postoperative are shown in Figures 5A and 5B, respectively. The density of vessels was significantly higher in the eyes treated with GH-MMC than in those treated with MMC alone (Table 2). 

Figure 5

 

Representative photomicrographs of vessel distribution in sections obtained from the eyes treated with GH-MMC (A) and those treated with MMC alone (B). Scale bars: 100 μm.

No significant difference was found in the density of PCNA-positive cells between the eyes treated with GH-MMC and those treated with MMC alone (Table 2). 

Discussion

The findings of this current study demonstrated that implantation of the MMC-loaded GH had almost the same effects on IOP reduction and filtration bleb formation as the 5-minute placement of MMC in a canine filtration surgery model. It is noticeable that its implantation also kept collagen density and the numbers of vessels and fibroblasts at a higher level than the conventional MMC application. In other words, the MMC-loaded GH had a comparable efficacy with less toxicity to the conjunctiva as a new DDS in canine models of glaucoma surgery. In addition, it might be reasonable that no significant differences were found in the densities of PCNA-positive cells, which reflect cell proliferation between the two groups, because the mechanism for maintaining bleb formation in each group might be similar. 

As in our previous study,²⁰ scleral trephining with peripheral iridectomy was chosen in this present study as a simple filtration surgery in canine eyes. Moreover, the effects of this DDS on the glaucoma surgery model were evaluated in a manner similar to that in the previous research.²⁰ According to another report²³ that included an in vivo release of basic FGF (bFGF), approximately 50% of the bFGF was released during the initial 3 days, with 30% being released during the following 11 days. Supposing that MMC might be released from the GH at a similar speed, approximately 17% of 0.012 mg, 2 μg MMC should be released as the sum per day for the initial 3 days in the GH-MMC group, whereas 0.1 mL 0.04% MMC (0.4 mg/mL) solution, which included 40 μg MMC, was exposed for 5 minutes in the MMC group. The ratio of MMC amounts exposed in the GH-MMC and MMC groups could probably be estimated to be lower than 1:20. In the GH-MMC group, the exposed concentration for the initial 3 days was 2 μg MMC in 0.0375 mL (0.0375 g) solution, 0.054 mg/g MMC per day, which was higher than 0.04 mg/g, the minimum concentration needed for inhibiting conjunctival fibroblast proliferation in vitro.²⁴ Taken together, the amount of MMC released from the GH in the present study should be effective for suppressing scar formation after glaucoma surgery and should induce less toxicity to the conjunctiva. 

Recently, there have been a few reports on the application of new DDSs containing MMC for glaucoma surgery. The surgical outcomes and wound-healing reaction after trabeculectomy using MMC-soaked biodegradable collagen matrix (Ologen; Optous, Roseville, CA, USA) was assessed on 30 eyes with glaucoma for 12 months.¹⁰ The results showed that this DDS generated encapsulated blebs at a more rapid pace in larger amounts compared with conventional trabeculectomy, yet comparatively stable IOP was maintained. Another study investigated the implantation of semicircular disks of poly(2-hydroxyethyl methacrylate) containing MMC, attached to an Ahmed glaucoma valve plate, in in vitro and in vivo rabbit models.⁸ The in vitro study revealed that this DDS released MMC over a period of 1 to 2 weeks, and in vivo histologic analysis demonstrated a significant reduction in inflammatory reaction and fibrosis in the resulting bleb. Thus far, the effects of this DDS on IOP reduction and bleb formation after glaucoma surgery have yet to be evaluated. In another report, a biodegradable thermo-sensitive hydrogel loaded with MMC for glaucoma surgery was evaluated both in vitro and in vivo.¹¹ The in vitro study showed that MMC was released from this DDS for up to 16 days, with 57% of the MMC being released in the first day. The in vivo study (using rabbits) indicated that this hydrogel loaded with 0.1 mg/mL MMC led to a functional bleb with prolonged bleb survival. The scar formation, new collagen deposition, and myofibroblast generation appeared to be reduced on histologic and immunohistochemical examinations in the eyes treated with the MMC-loaded hydrogel compared with the eyes treated with a conventional application of MMC, although no significant difference in IOP reduction was found between them for 28 days postoperative. The results of that study appear to be similar to those of our current research, although the experimental animals (rabbits and beagles, respectively) and glaucoma surgery models were different from one another. 

It should be noted that the present study had several limitations. First, the effect of this DDS might be different in glaucoma surgery performed on human eyes. Thus, the effect and toxicity of the DDS applied in this study should be verified in primates in the future. Moreover, longer-term observation of the effects and toxicities of this DDS might be needed. Furthermore, the most suitable dosage of MMC for use in this DDS for glaucoma surgery should be investigated in detail. 

In conclusion, our results suggested that implanting MMC-loaded GH as a new DDS was comparably effective for maintaining filtration bleb formation and less toxic to conjunctiva than the conventional application of MMC for glaucoma surgery. This DDS appears to be worthy of further investigation to improve the outcomes of glaucoma surgery. 

Acknowledgments

The authors thank John Bush for reviewing the manuscript. 

Supported by Japanese Ministry of Education Grant 24592684 (SK). 

Disclosure: S. Kojima, None; T. Sugiyama, None; S. Takai, None; D. Jin, None; M. Ueki, None; H. Oku, None; Y. Tabata, None; T. Ikeda, None