The Effect Of Sirolimus Eluting Stent On Tracheal Stenosis After Stenting

Tracheal stenosis followed by stent implanting is a difficult clinical problem,the treatments for in-stent granulation hyperplasia include endoscopic interventional therapy and drugs therapy,the former includes laser,high frequency electrocogulation et al[1],while repeated interventional therapy costs high hospitalization expenses and may increase burdens of patients' economy and psychology.Drugs therapy includes systemic and local treatment,some study showed that systemic application of dexamethasone,immune inhibitor could inhibit the growth of tracheal granulation tissue[2][3],while the side effects of systemic application were severe.Local application of drugs can make drugs specifically play a role in the target lesions and avoid the systemic effects.According to the current studys,inhaled budesonide or local application of mitomycin C,paclitaxel could effectively control the hyperplasia of granulation[4]?[7],but local application of drugs can't continue to inhibit the growth of granulation tissue due to its short working time.In recent years,the advent of drug eluting stent has resolved this question,the combination of drugs and polymer achieves the goal of sustained release by coating stent.Previous studies have demonstrated that the combined therapy of mitomycin C,cisplatin and dexamethasone drug-eluting stent was successfully applied on animal models,in which the drug sustained releasing more than 1 month,and inhibited granulation hyper:plasiL[8]?[10]The mechanism of granulation hyperplasia includes local tissue compression,inflammation,vascular epithelial excessive expression et al;Among those immunosuppressant can inhibit mammals rapamycin target protein(mTOR)pathway which control granulation hyperplasiac[11].Sirolimus,as a kind of immunosuppressant,belongs to the macrolides antibiotic which carries out various functions including anti-inflammation,antiproliferative and anti-rejection.The results from animals study showed that sirolimus could inhibit mammals rapamycin target protein(mTOR)pathway by suppressing inflammatory cascade reaction,thus drug was able to control the growth of granulation tissue and repair damage healing[12][13].The data from clinical research showed systemic application of sirolimus had an inhibitory effect on granulation tissue formation among these patients who received stent implantation after lung transplantation[2].Domestic scholars found that rapamycin-coated metallic stents were effective on relieving airway obstruction in children with congenital tracheal stenosis[14].So sirolimus may have a potential inhibitory effect on granulation hyperplasia.Poly(lactic-co-glycolic acid)(PLGA)has been well recognized for its suitability in drug delivery and possesses good biocompatibility,it doesn't cause serious immunoreaction and cytotoxic reaction,and metabolizes completely into water and carbon dioxide after completing drug release,leaving the bare-metal stent in situ.Research has confirmed that PLGA never interacts with sirolimus[15]and is the first choice of drug loading platform.Among different stent materials,the stent type of clinic commonly is metallic stent,metallic stent can not only fit small-lumen trachea and be dilated as the infant grows,but also be simple to insert[16].While metal alloy stent is better than bare-metal stent on increasing the radial strength,improving the radiation impermeability,and its biological inert is stronger[17][18].Among of metal alloy stent nickel titanium alloy stent we selected in the study has unique shape memory function,good dynamic expansivity,ability of resistance to deformation,strong mechanical property,corrosion resistant,and don't affect drainage of sputum[19].The common methods for stent coating include dipping coating and spraying painting,Yongpeng Chen et al from the third military medical university found that the drug loading of stents prepared by dipping coating was higher than spray painting when comparing the two methods with dexamethasone eluting stent,and the spraying painting-prepared stents may result in uneven distribution of drugs[20],thus dipping coating is more suitable for preparation of drug stent.The common animals to choose for experimental subjects include pigs,dogs,monkeys,rabbits et al,among these dogs and pigs are ideal models for adults airway diseases due to that their trachea cavity is wider and similar to adults,but they are relatively expensive,not easy to control,handle,fix,these limit their application in the experimental study.Monkeys' physiological anatomy resembles person,while study about monkeys is few because of its expensive costs,hard feeding,difficult degree of experiment and so on.Rabbits are cheap,easy to raise and operate safely,the study found the general trachea size of 4 years old children was similar to the trachea size of 4 months rabbits[21],so we choosed adult rabbit trachea to simulate children trachea.Overall averages for airway size at the level of the cricoid are 5.81 mm ventral-dorsal,5.41 mm lateral[22],7?8.5cm in length[23],the trachea stent of 6mm diameter,30mm length is appropriate according to the principle that the diameter of stent should be 1.2?1.3 times of trachea diameter.4 Nitinol stent as platform,PLGA as drug-loaded materials,dipping coating as method,was selected,we prepared the trachea drug stent with sirolimus.The rabbit trachea was incised annularly and separately placed sirolimus eluting stent or bare-metal stent,the laboratory reference is provided for clinical application of drug eluting stent by comparative observing the effect on trachea in-stent stenosis from slow-released drug.Objectives1.To prepare the sirolimus eluting stent by dip-coating method and detect its drug concentration,distribution of durg coating.2.To explore the safety of sirolimus eluting stent on the application of animal model by inserting drug eluting stents with tracheotomy.3.To explore the efficacy of sirolimus eluting stent on in-stent granulation hyperplasia and inflammation response by inserting respectively drug stents,bare-metal stents into animals trachea of experimental,control group.Methods1.The preparation of sirolimus eluting stent:The bare-metal stents are cleaned according to relative literatures[24],the preliminaryexperiment showed the ratio of sirolimus and PLGA at 0.04:0.4(g)by weight is the longest in releasing time which could release more than one month.Blended solution containingyPLGA and sirolimus was prepared to fabricate drug eluting stent by dip-coating.Scanning electron microscopy(SEM)is used to get the thickness and the distribution of drug coating on the surface of stent.The drug concentration of stent is detected by high performance liquid chromatography(HPLC).2.The study about safety and efficacy of sirolimus eluting stent on the application of animal model:Sixteen New Zealand white rabbits were randomly divided into the sirolimus eluting stent group(n=8)and the bare-metal stent group(n=8).The rabbit trachea was incised annularly and separately placed sirolimus eluting stent or bare-metal stent,and the tracheal was then closed.The granulation hyperplasia in trachea in-stent was observed by ultrathin bronchoscope at the 2nd and 4th week after operation.The severity of tracheal stenosis under endoscope was estimated by percentage stenosis of the cross-sectional area of the trachea,and the cross-sectional area of the trachea which was less than 25%was defined as mild,25%?50%as moderate,50%?75%as severe,more than 75%as very severe,respectively.All animals were euthanized at the 4th week.The specimens of trachea were obtained and observed under light microscope after HE staining.Results1.The preparation of sirolimus eluting stent:The attachments of stent surface were removed by ultrasonic cleaning method with dichloromethane,and no damage was found on the stent under SEM.We successfully prepared the sirolimus-eluting trachea stent with dipping method,and stent coating was obtained with the thickness of about 4?5?m.The SEM showed that the distribution of drug on stent was uniform.We dissolved the drug stent with organic solvent and detected the sirolimus concentration of organic solvent by HPLC method,the final dosage of sirolimus per stent is 80.5?g.2.The effect of sirolimus eluting stent on in-stent granulation hyperplasia:Each group had one animal death in experimental process,the other animals survived to the end and all appeared different degree of trachea stenosis.The ultrafine bronchoscope showed mild granulation hyperplasia of in-stent was observed at the 2nd week of postoperation in control group,and partial animals had heavily mucus retention,while in the sirolimus eluting stent group no visible granulation tissue formed and a small number of mucus retention could be seen.At the 4th week after stent placement,mild-to-moderate granulation tissue formed in the control group,even completely obstructed in individual case,the top or lower edge of stent had visible granulation hyperplasia.Mild granulation tissue was seen in the DES group,in which the top edge of stent was the most common.Stent migration,fracture was not found.The trachea specimens in sites of in-stent granulation hyperplasia and sutured region were obtained,we measured the thickness of granuation under the 400X perspective of optical microscope by Image J software.The degree of inflammation was devided inito 5 kinds,in 5 randomly 400X perspectives.Cases showing no inflammation were scored 0;mild inflammation of less than 10%of the affected area,1+;less than 30%of cellular infiltrates in the affected area,2+;intense inflammation(30%-60%),3+;and diffuse inflammatory infiltrates of more than 60%of the affected area,4+[25].The data for statistical analysis showed that less thickness(?m)of in-stent granulation tissue in the experimental group(1577.529+507.971)was detected compared to that in the control group(812.945+235.893)(t=3.612,P=0.004),and the inflammation response induced in experimental group(mean rank 4.93)was weaker than that in control group(mean rank 10.07)(Mann-Whitney U=6.500,P=0.014),and the stronger the inflammation was,the more obvious granulation tissue hyperplasia becomed(the correlation coefficient r=0.809,P=0.000),the difference was statistically significant.Our study determined that granulation hyperplasia didn't exist in the sutured region,mild inflammation was found.Conclusions:1.The dip-coating method for making drug stent is simple which can make the drug uniform distribution in the surface of the stent.We successfully prepared the drug stents which meet the requirement of experiment by dip-coating method.2.The homemade drug stents are proved to be safe and effective novel trachea drug-eluting stents on animal models,it highlights the potential value of clinical application,This study may be the laboratory basis for the future stent design.3.Sirolimus eluting stent can inhibit the granulation hyperplasia and reduce inflammation response after trachea stent implantation when compared to bare-metal stent.4.The thickness of in-stent granulation hyperplasia is highly related to the degree of inflammation.5.Granulation hyperplasia or trachea stenosis does't exist in the sutured region,only mild inflammation response can be found.