| Tracheal stenosis is caused by infections,tumors,trauma,endotracheal intubation,congenital disorders and other diseases,with clinical manifestation of cough,sputum,dyspnea,and even respiratory failure,and life-threatening.Tracheal stenosis is one of common severe diseases of respiratory critical care medicine emergency.,which may affected ventilation function,caused breathing difficulties,and then developed into the respiratory failure,even tend to death.With the continuous development of respiratory endoscopic interventional treatment technology,tracheal stent implantation technology which continues to mature,more interdisciplinary communication,and the combination of freezing,thermal ablation and other technologies,can play a better role in the treatment of tracheal stenosis.Patients with tracheal stenosis can rapidly relieve dyspnea after tracheal stent implantation and improve clinical symptoms.Tracheal stent implantation is one of the important methods for the treatment of tracheal stenosis.Stent implantation may result in a wide range of complications,such as in stent restenosis,cough,infection,bleeding,which hinder the tracheal stents widely used.Following the deep research in the pathogenesis of tracheal stenosis,found that the formation of tracheal stenosis with recurrence is actually the airway mucosa damaged triggered a series of abnormal fibrin hyperplasia repair responses,in which the key role is the abnormal activation of fibroblasts.Therefore,how to reduce the granulation tissue hyperplasia become the research focus.The local application of antiproliferation drugs to inhibit the excessive proliferation of tracheal fibroblasts is a way to solve this problem.Therefore,we need to design a special stent,which can support the expansion of trachea enough to keep trachea unobstructed,can inhibit the proliferation of granulation tissue after stent implantation,and prevent granulation hyperplasy of restenosis,so as to expand the application of tracheal stent in clinic.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 and clinical research 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.Sirolimus may have a potential inhibitory effect on granulation hyperplasia.Objectives1.To explore the preparation of sirolimus eluting stent.2.To explore the pharmacokinetics of sirolimus eluting stent in vitro.3.To explore the safety and efficacy of sirolimus eluting stent on the application of animal model by inserting drug eluting stents with tracheotomy.Methods1.The preparation of sirolimus eluting stent:Bare Nitinol tracheal stents were carefully cleaned using an ultrasonic cleaning method with dichloromethane and distilled water in sequence.The cleaned stents were kept under a fume cupboard for 24 hours to evaporate the residual water.The stents were dipped vertically into a coating solution prepared by different concentration ratio of sirolimus in 20ml of dichloromethane.Samples were kept at 3 70C while being constantly for one day.Repeat the above steps.Finally,the DESs were placed in an oven overnight at 37? to remove any solvent.2.The pharmacokinetics of sirolimus eluting stent in vitro:Coated stents were submersed in 10 ml of PBS at pH 7.4.PBS.The samples were kept at 37? while being constantly agitated at 75 revolution/min in an incubator shaker.Every two days,the incubation medium was removed for analysis,and completely replaced with fresh medium.These results were used to plot the cumulative release over time.3.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.All animals were euthanized at the 4th week.The specimens of trachea were obtained and observed under light microscope after HE staining.Results1.Ultrasonic cleaning method with dichloromethane can remove the impurity of stents.No damage was found on the stent under scanning electron microscopy.The scanning electron microscopy were used to observe stent morphology,It was indicated that not only the coating was very smooth and uniform,but also the coating had not any webbings between stents.We successfully prepared the sirolimus-eluting trachea stent with dipping method,and stent coating was obtained with the thickness of about 4?5um.2.The Drug loading for different drug loading conditions are distinct.The proportion of sirolimus/PLGA stents of 1:10 loaded the most amount of drug,so the optimal proportion of sirolimus/PLGA is 1:10;We also attempted to increase drug loading by increasing the ratio of sirolimus in the coating solution,increasing the ratio of sirolimus/PLGA concentration(sirolimus/PLGA I:5)cannot improve stent drug loadings,However,increasing the ratio did not improve drug loading.It suggest that the prompt stent drug-polymer interactions exist saturation effect.Concentration of sirolimus in PBS solution were detected by enzyme immunoassay amplification every two days,it continuous detection for 42 days.The amount of sirolimus released from a stent per day was about 80.07±2.26ug and exhibited a sustained,steady release.In vitro experiments showed that there was measurable drug release for more than 42 days,with about 50%of the loaded drug released during the first 2 weeks,about 70%during the first 28 days,80%during the first 42 days.We assumed that all of the loaded sirolimus would be released More than 6 weeks.Additionally,drug release in DES in vitro were mainly based on the drug diffusion and degradation of the polymer,there was a quick release in first 10days and subsequent a slow release period of drugs in DES in vitro.Thus,the results were satisfactory,and could meet the needs to inhibit granulation formation.3.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 data for statistical analysis showed that less thickness(?m)of in-stent granulation tissue in the experimental group(812.945±235.893)was detected compared to that in the control group(1577.529±507.971)(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.1ur study determined that granulation hyperplasia didn't exist in the sutured region,mild inflammation was found.Conclusions1.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 best ratio of sirolimus/PLGA coating is 1:10,which enables the stent carry most drugs.The drug loading of stent did not increase when continue to increase the concentration of sirolimus in solution,suggesting a saturation effect of coating carrying drugs.Perform an initial evaluation of the sirolimus-coated stent pharmacokinetics in vitro.The amount of sirolimus released from a stent are safe in the administration scope,no correlation of drug poisoning.3.The thickness of in-stent granulation hyperplasia is highly related to the degree of inflammation.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,Sirolimus eluting stent can inhibit the granulation hyperplasia and reduce inflammation response after trachea stent implantation when compared to bare-metal stent. |
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