Study On The Effect And Mechanism Of Mitomycin C And Chitosan On Intraarticular Scar Adhesion After Knee Surgery

Intraarticular scar adhesion of knee is one of common complications after knee surgery, which often affects the longterm postoperative effect. The formation of restrictive scar adhesions can alter the knee functions, lead to loss of flexion and extension, stiffness and other functional impairments. Recently, mitomycin C (MMC) and chitosan have been reported to prevent scar adhesion in clinical and experimental studies. However, few studies were reported on mechanism of MMC and chitosan in reducing knee intraarticular scar adhesion. We illustrated the effect of MMC and chitosan on inhibiting human fibroblast proliferation and inducing apoptosis in vitro, and evaluated the effect of these drugs on reducing intraarticular scar adhesion in a rabbit model. The present study could identify the mechanism of MMC and chitosan on preventing knee intraaticular scar adhesion, provide theoretical basis and new therapic target in preventing intraarticular adhesion after knee surgery for clinical, and find a simple and effective method to prevent intraarticular scar tissue formation after knee surgery. Chapter One Experimental study of the effect of mitomycin C and chitosan on inhibiting fibroblasts proliferationObjective To evaluate the effect of mitomycin C and chitosan on inhibiting human fibroblasts proliferation.Methods Fibroblasts were cultured in vitro and treated with various concentrations of mitomycin C and chitosan. Fibroblasts were collected at1,2,3,5days after treated with the drugs for observing morphological changes under inverted microscope. MTT testing method was used to evaluate the effect of mitomycin C and chitosan on inhibiting fibroblasts proliferation.Results1. Fibroblasts exhibited different morphological changes after treated with different concentrations of mitomycin C and chitosan. With the concentration of drugs increased, the growth of fibroblasts were obviously inhibited, the rate of inhibition significantly increased and the number of fibroblast decreased dramatically.2. As time went on, the inhibition of fibroblasts gradually strengthened and rate of inhibition gradually increased after treated with the specific concentration of mitomycin C and chitosan.Conclusions1. After treated with different concentration of mitomycin C and chitosan in vitro, there was significant changes in morphology of human fibroblasts and growth characteristics. The results of morphology and MTT method showed that mitomycin C and chitosan could inhibit proliferation of fibroblast.2. With the concentration of mitomycin C and chitosan increased, the inhibition of mitomycin C and chitosan on fibroblasts proliferation gradually strengthened.3. As time went on, the inhibition of mitomycin C and chitosan on fibroblasts proliferation gradually strengthened. Chapter Two Study of the mechanism of mitomycin C and chitosan on inducing fibroblasts apoptosisObjective To investigate mechanism of human fibroblast apoptosis induced by mitomycin C and chitosan.Methods Fibroblasts were cultured in vitro and treated with various concentrations of mitomycin C and chitosan. Fibroblast apoptosis was observed by Hoechst33342staining and cell apoptosis rate was determined through Annexin V-PI methodd. The expression of related apoptotic proteins such as active caspase-3, Bax, CHOP and Bcl-2were detected by western blot.Results1. Hoechst33342staining showed the number of apoptotic nucleus of fibroblasts in mitomycin C-treated groups were more than that of control group. Moreover,0.8mg/ml mitomycin C group exhibited the maximum of apoptotic nucleus.The number of apoptotic nucleus of fibroblasts in chitosan-treated groups were more than that of control group. Moreover,10mg/ml chitosan group exhibited the maximum of apoptotic nucleus.2. The results of Annexin V-PI double staining showed that the apoptosis rate in mitomycin C-treated groups were significantly higher than that of control group.Moreover, the apoptosis rate of0.8mg/ml mitomycin C group was significantly higher than those of other groups. The apoptosis rate in chitosan-treated groups were significantly higher than that of control group.Moreover, the apoptosis rate of10mg/ml chitosan was higher than those of other groups.3. The results of western-blot showed the protein expression of active-caspase-3, Bax and CHOP gradually increased with the concentration of mitomycin C increased. In contrast, the protein expression of Bcl-2was continuously decreased. The protein expression of active-caspase-3, Bax and CHOP in chitosan groups gradually increased with the concentration of chitosan increased. However, the protein expression of Bcl-2was also decreased.Conclusions1.After human fibroblasts were treated with different concentrations of mitomycin C and chitosan in vitro, the results of Hoechst33342staining and Annexin V/PI double staining showed mitomycin C and chitosan could induce fibroblasts apoptosis.2. With the concentration of mitomycin C and chitosan increased, the rate of fibroblast apoptosis gradually increased and exhibited the dose-effective manner.3. The result showed that mitomycin C and chitosan could induce human fibroblast apoptosis through many apoptosis pathways such as the mitochondrial pathway, death receptor damage, endoplasmic reticulum stress. Chapter Three Experimental study of the preventive effects of mitomycin C and chitosan on intraarticular adhesion after knee surgery in rabbitsObjective To observe and compare the preventive effects of mitomycinC and chitosan on intraarticular adhesion after knee surgery in rabbit.Methods48New-Zealand rabbits were used to establish animal model of scar adhesion after knee surgery.The animals were randomly divided into three groups:mitomycin C(MMC), chitosan and control group,16rabbits in each group. Gross observation, histopathological examination, hydroxyproline contents of scar and fibroblast counts were performed to evaluate intraarticular adhesion4weeks postoperatively.Results All the experimental animals had good recovery, and there was no case of wound infection, necrosis or mortality. The results showed as follows:1. Mild membrane-like fibrosis was found in MMC group and moderate intraarticular adhesion was found in chitosan group, while there was large-size compact fibrous tissue adhesion in control group.2. Hydroxyproline contents of MMC and chitosan groups were lower than that of control group (P<0.05). Moreover, the content of MMC group was lower than that of chitosan group (P<0.05).3. There was only thin and membrane-like scar tissue with few fibroblasts in MMC group. Moderate scar tissue and the quantity of fibroblasts were found in chitosan group. In control group, thick and marked scar tissue mixed with large mount of fibroblasts was found in the decorticated area.4. The fibroblast counts in MMC and Chitosan groups were significantly lower than that of control group (P<0.05).The fibroblast counts in MMC group was also lower than that of control group (P<0.05).Conclusions1. The animal model of intraarticualr scar adhesion after knee surgery was successfully established.2. Topical application of MMC and Chitosan could prevent the formation of scar adhesion after knee surgery in rabbits by inhibiting the proliferation of fibroblasts and decreasing the formation of collagen.3. There was no case of wound infection, necrosis or mortality and no obvious complication was found by histopathological examination. The reason maybe include low concentration of MMC and chitosan, short time in contract with tissue and little absorption of these drugs.