The Experimental Study Of Colonic Anastomosis With Biodegradable Doxycyline-eluting Polylactide Stent

Part â… The drug release in vitro for biodegradable doxycycline-eluting polylactide stent measured by HPLCObjective To investigate the efficacy of drug release for biodegradable doxycycline-eluting polylactide stent by HPLC.Methods The biodegradable doxycycline-eluting polylactide stent was placed in105ml phosphate buffered saline solution for2weeks.50ml of phosphate-buffered solution was changed daily and the daily amount of doxycycline released was measured by high-performance liquid chromatography. The concentration of doxycycline was assayed on a C18column with a mobile phase consisting of methanol-acetonnitrile-water(55:25:20) at a flow rate of1ml/min and detected at374nm.Result A linearity was obstained from0.5to4ug/ml of doxycycline in water with a good correlation(r=0.9862).The total amount of doxycycline released was about11.595ug over two weeks. The releasing rate was77.24%on the first day,87.08%on the third day and97.4%on the seventh day. The drug release was stopped on the thirteenth day.Conclusion The pattern of drug release of biodegradable doxycycline-eluting polylactide stent in vitro was in accordance with the expectancy of experimental design. The release rate was higer during first three days. Part â…¡Cytotoxicity evaluation of biodegradable doxycycline-eluting stent for fibroblast in vitro studyObjective To evaluate the effect of biodegradable doxycycline-eluting stent on the proliferation and apoptosis of fibroblast.Methods Extract solution of biodegradable doxycycline-eluting stent of72hours was freshly prepared. Fibroblast was incubated with extract solution of biodegradable doxycycline-eluting stent for48hours. Morphological observation of fibroblast was performed through microscope. Cytotoxicity was evaluated by MTT assay and relative growth rate was calculated and graded. The cell cycle and the apoptosis rate of fibroblast were analysed by flow cytometry. Cell culture medium served as negative controls.Results No obvious difference of morphology and stucture of fibroblast was observed by microscope between experimental group and the control group after48hours incubation. The OD value of fibroblast in experimental group was slightly decreased without statistic difference. The relative growth rate was96.5%and cytotoxicity degree was â… . The percentage of G2phase cell and apoptosis rate were analysed. There was no statistic difference between the experimental group and control group.Conclusion biodegradable doxycycline-eluting stent has no obvious cytotoxicity effect on the activity and growth of fibroblast, which provide the theoretical basis for the following animal studies. Partâ…¢The effect of a biodegradable doxycycline-eluting stent on wound healing of the colonic anastomosis in porcine modelObjective To evaluate the effect of a biodegradable doxycycline-eluting polylactide stent on colonic anastomosis by observing the healing process after stent placement in a porcine model.Methods48Bama minipigs were randomized to three groups with different anastomosis methods in this experiment:doxycycline-eluting stent group(DS); polylactide stent group(PS) and hand-sewn group(HS). Wound healing of the stomas were observed on day3,7,14and30postoperatively in each group. After anastomotic tissue harvested, Bursting pressure and hydroxyproline contents were measured and compared between different groups. HE&Masson staining, immunohistochemical staining of TGF-(3l, bFGF, a-SMA and CD31were examined. Moreover, the relative expression of MMP-2and MMP-9mRNA were performed and compared.Result All operation were accomplished successfully. There were two cases of death due to excessive anesthesia(DS) and colonic leakage(HS) respectively. Bursting pressure of DS&PS was significantly higher than HS on day3and day7. Healing status between different groups was similar, there was no significant difference in hydroxyproline content and immunochemistry of TGF-(31, bFGF, a-SMA and CD31. The relative expression of MMP-2mRNA of DS was significantly decreased with comparision to PS, however the expression of MMP-9mRNA was similar between DS and PS.Conclusion No obvious negative influence of biodegradable doxycycline-eluting polylactide stent was observed in vivo. It was safe and effective on the colonic anastomosis. Local delivery of doxycycline via a biodegradable stent has the potential to improve wound healing of colonic anastomosis in porcine model.