| Background:The common reason for laryngotracheal stenosis is mechanical trauma such as cartilage fracture, airway laceration, displacement or deformity after laryngotracheal trauma; and iatrogenic injury such as tracheal intubation, tracheotomy, laryngeal radiotherapy, operation and so on. Subglottic stenosis and tracheal stenosis is the most common in clinic, and current treatments include surgery, laser therapy, stent expansion, drugs and so on. Tracheal reconstruction, cricoidectomy, CO2and Nd:YAG laser therapies are the frequent operation methods. Stents are usually needed in prevention of restenosis after operation and obstruction in airway because of laryngeal-tracheal malignant tumors. The current feasible stents causes airway injuries as a result of bad compliances with airway anatomical structure, cough because of stimulation to laryngeal mucosa, migration, granulation growth, re-stricture after extubation etc. These complications haven’t got resolved completely. As for benign laryngeal-tracheal stenosis, it will restenosis due to fibrous tissues proliferation in one half year to one year after stent drawn out. Granulation formation is the most common complication after placing stents. The function of anti-tumor, anti-scarring, reducing granulation proliferation and preventing tissues adhesion of MMC(mitomycin C) has been recognized and widely used in many clinical fields in recent years.MMC was first discovered in1956which was one kind of broad-spectrum antineoplastic antibiotics separated and extracted from head-like Streptomyces culture solution, its mechanism is depolymerizing DNA in cell, impeding reproduction, thereby inhibiting cell mitosis (including tumor cells and fibroblast). As a broad-spectrum anticancer drug, MMC is used as systemic chemotherapy drugs, such as in breast, lung, pancreas and colorectal cancer treatment. MMC has the dual role of antitumor and inhibition of proliferation, and as one kind of alkylating agent, its main mechanism is the formation of the double helix of DNA cross-linking, destruction of DNA structure and function, inhibition of DNA replication in both proliferative and quiescent stage, and thus can be used to prevent angiogenesis and scar formation. MMC can down regulate the expression of cyclin D1and up regulate caspase-3of fibroblast in vitro. Caspase-3is known as the death protease, its increase will activate the apoptotic cascade reaction and make cells turn into irreversible process of apoptosis, and induce apoptosis. MMC prevents scar formation through inhibiting proliferation, inducing apoptosis and reducing fibroblast, and in prevention of adhesion after vocal cord operations and stenosis after tracheal operations, MMC can play a role through those pathways we talked above.In ENT (Ear,Nose,Throat)clinic, MMC has certain effect in prevention of laryngeal-tracheal and nasal stenosis. But all the studies are one-time drug administration that using one piece of cotton soaked with MMC(0.4mg/Ml) applied locally in the wound surface for4-5minutes, and the results show that the problem of adhesion can’t be completely resolved. It is reported that Poly L-lactide-co-glycolide(PLGA) nanoparticles could be used as laryngeal slow-release drug carrier in murine vocal fold injection model, the results show that PLGA loaded with Texas Red-dextran (NPTR), hepatocyte growth factor (NPHGF), and bovine serum albumin (NPBSA)can slowly release over12-14days in vitro, and in vivo presence of NPTR occurred up to7days compared to1day for Texas Red control. In addition, NPHGF ameliorated transforming growth factor-beta induced procollagen in vitro in3T3fibroblast cells, and the results demonstrate the potential utility of nanoparticle encapsulation as an effective method for long-term delivery of specific drugs and biologically active substances to the larynx. Temperature-sensitive PLA hydrogel which is biodegradable and has good biocompatibility with tissue can change with ambient temperature into reversible phase. Taking advantage of PLA phase change characteristic, it can be served as filling material as a result of tissue defect or drug slow-release carrier. PLA hydrogel is liquid in refrigerated condition and colloid at room temperature which can be applied in laryngeal-tracheal diseases as drug delivery carrier. When temperature sensitive PLA gel is injected into the body, it will be curdled in situ at body temperature. We can mix the required doses of drugs with PLA gel, which can be injected into disease affected site and form gel in short time. This in-situ formed hydrogels which can be fixed in certain parts of body have good solid combination with the surrounding tissue, it can precisely release wrapped drugs in lesions and effectively control the extent of drug delivery, thereby reduce systemic adverse reactions.DSP(dexamethasone sodium phosphate) as one kind of glucocorticoid has significant effect on inflammation which can be caused by various factors such as physics, chemistry, biology, immunology and so on. In early time of inflammation, it can relieve leakage, edema, capillary expansion, leukocyte infiltration, phagocytic reaction. In late time, it can inhibit hyperplasia of capillary and fibroblasts, delay granulation tissue forming, prevent adhesion and scar formation. DSP has a little side reaction, its price is cheap and it can be used to inhibit formation of fibrous tissues. This experiment aims to use PLA as MMC and DSP slow-release carrier to study the effect of MMC/PLA and DSP/PLA on inhibiting fibroplasias of trachea in animal. And this maybe provide experimental basis for studying PLGA scaffold slow-delivery system for treating laryngotracheal stenosis in the future.Objectives:To compare the effect of slow-release mitomycin C(MMC) and dexamethasone sodium phosphate(DSP) with poly lactic acid (PLA) hydrogels on prevention of tracheal wall fibroplasia.Methods:In vitro release study,1mg MMC and2mg DSP mixed with1ml PLA hydrogels released in phosphate buffered saline(PBS) at constant temperature of37℃and rotational speed of120round per minute (rpm), the cumulative concentrations were determined by ultraviolet spectrophotometry. In vivo study, the rabbit model of tracheal wall fibroplasia was made through scratching the tracheal wall. Forty-two rabbits were divided into7groups averagely, and0.2ml PLA hydrogels contained different doses of MMC and DSP mixed with spongia gelatinosa composite were first put in semicircular silicone tubes and then they were placed on the wound surface of scratched tracheal wall. There were three MMC groups (0.1mg,0.2mg,0.4mg), three DSP groups (1mg,2mg and3mg) and one control group. And then the concentration of0.4mg MMC group in plasma of1st,3rd,7th,10th,14th and21st day after operation was determined by mass spectrometry(MS). Each animal was killed by air embolism4weeks later. All the specimens were fixed with formalin, embedded with paraffin, sliced and stained with hematoxylin and eosin(H&E), and then observed with light microscope. The thickness of fibroplasia was calculated by the vertical distances from the highest point of hyperplasia tissue to the outer surface of tracheal wall. The data were analyzed by SPSS13.0with One-way ANOVA LSD test.Results:In vitro study, PLA hydrogels with1mg MMC and2mg DSP could release drug over35days and28days respectively. And in vivo study, these two kinds of PLA hydrogels hadn’t degraded completely after4weeks. The tracheal wall fibroplasia in control group was thicker than that in both DSP and MMC groups except0.1mg MMC group. Among MMC groups,0.4mg MMC group had the best effect in inhibiting fibroplasia. While in DSP groups, the thickness of fibroplasia had no difference among three groups; and the difference of fibroplasia thickness was not significant between DSP groups and0.4mg MMC group. The leukocyte count on the1st,7th and14th day after operation had no significant difference.Conclusion:MMC and DSP PLA hydrogels can release drug over4weeks either in vitro or in vivo. Both MMC/PLA and DSP/PLA hydrogels could inhibit fibroplasia of the tracheal wall and can be used as slow-release administration in prevention of fibroplasia, which provided experimental basis for studying PLGA scaffold slow-delivery system for treating laryngotracheal stenosis in the future. |
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