Inhibitory Effect of Mitomycin C (MMC) on the Proliferation of Human Corneal Keratocyte

PURPOSE: To evaluate the anti-proliferative effect of mitomycin C (MMC) on human corneal keratocyte, and to investigate the cellular morphology of keratocyte according to the concentration and exposure time in vitro. METHODS: Human corneal keratocytes using endothelium-free explant method were exposed to 0.005%, 0.01%, and 0.05% concentration of MMC for 3, 5, and 10 minutes. MTT based colorimetric assay was performed to assess the inhibition of cellular proliferation, and cellular morphology was evaluated by inverted phase-contrast light microscope and electron microscope. RESULTS: Use of higher concentration MMC and prolongation of exposure time resulted in greater inhibitory effect on cellular proliferation. When exposed to 0.005% MMC for 3, 5 and 10 minutes, the survival rate of keratocyte was 100%, 95.7% and 74.0% respectively. At 0.01% MMC, the survival rate was 98.6%, 92.9%, and 66.9%. At 0.05% MMC, it was 74.0%, 73.4%, and 38.8%. Exposure to the highest concentration (0.05%) among the 3 preparations for 3 or 5 minutes showed significant inhibition of keratocyte proliferation (p<0.05), and when exposed for 10 minutes, all 3 preparations showed significant inhibition of keratocyte proliferation (p<0.05). Inverted phase-contrast light microscopy showed that human corneal keratocytes lost their adherence to the bottom of the dish and assumed round and swollen shape rather than spindle shape when exposed to higher concentration of MMC for a prolonged time. The damaged keratocytes showed the degenerative changes like cellular membrane disruption, disappearance of microvilli, enlargement of rough surfaced endoplasmic reticulum and mitochondria, and vacuole formation by electronic microscope. CONCLUSIONS: When MMC is applied to inhibit the proliferation of keratocytes involved in corneal wound healing, it seems to be a valuable application at least 0.05% concentration for 3 minutes. Further studies should be followed for the biological effect of MMC including drug toxicity associated with human corneal tissue in vivo.

Effect of Anti-inflammatory Mediator on the Proliferation of Human Corneal Keratocyte

PURPOSE: The purpose of this study was to evaluate the effect of anti-inflammatory mediators like dexamethasone, nordihyroguaratic acid (NDGA), and diclofenac sodium on proliferation of human corneal keratocytes, and to investigate the cellular morphology of keratocyte. METHODS: Human corneal keratocytes were exposed to 0.05, 0.1, 0.3, 0.8, and 1.0 mM concentration of each drug for period of 24, 48, and 72 hours. MTT based colorimetric assay was performed to assess the metabolic activity and inhibition of cellular proliferation. Cellular morphology was evaluated by inverted phase contrast micrograph and electron microscopy. RESULTS: The higher the concentration of inoculated each drugs was, the more the inhibitory effect of human keratocyte proliferation was found (P<0.05). NDGA, over 0.3 mM and diclofenac, more than 0.1 mM had significant more inhibitory effect on keratocyte proliferation compared with dexamethasone within 48 hours of exposure to each drug. With the concentration and exposure time of each drug, human corneal keratocytes were visible more rounded and swollen rather than spindle shape, and detached from the bottom of the dish. The damaged keratocytes had degenerative changes like cellular membrane disruption, microvilli disappearance, enlarged rough surfaced endoplasmic reticulum and mitochondria, vacuole formation and nuclear membrane damage by TEM. CONCLUSIONS: On basis of this study, the anti-inflammatory mediators such as NDGA and diclofenac sodium have less side effects and stronger inhibitory effects of human keratocyte proliferation than dexamethasone.

The Effect of Mitomycine C (MMC) on Inhibition of Pterygial Fibroblast Proliferation

The purpose of this study is to investigate that the biological effect of mitomycin C(MMC) in cellular metabolic activity and morphological change on the ptreygium fibroblast in vitro by MMC concentration and duration of exposure used clinically. Human pterygial fibroblasts were exposed for threeminute and five-minute to MMC 0.002%, 0.004%, 0.01%, 0.02%, 0.04%, and DMEM(control). MTT based colorimetric assay was performed to assess the metabilic activity, inhibition of fibroblast proliferation on the MMC concentration and exposure time. The higher the concentration of MMC, and longer the duration of exposure time, the absorbance of spectrometer are decreased. The metabolic activity of fibroblasts were inhibited by 50% at least only over MMC 0.02% for five-minute expoure time. In histological findings, the higher the concentration and longer the duration of MMC exposure time, the enlargement of many mitochondira and rough endoplasmic reticulum without nuclear damage were more distinctly appeared. Especially, the ptergial fibroblast has more severe cytoplasmic damage at a five-minute exposure to MMC 0.02% than a three-minute exposure to MMC 0.04%. For inhibition of fibroblast proliferation, in case of using MMC should be at least over 0.02% concentration for five-minute exposure time. Arthors think that the experimental and clinical research on the duration of MMC exposure time as well as the concentration MMC, should be need to evaluate the effect on inhibition of cellular proliferation.