季铵化壳聚糖-重组组织因子途径抑制物复合物对大鼠碾压撕脱皮瓣的影响

Effects of N-trimethyl chitosan-recombinant tissue factor pathway inhibitor complex on avulsion flap with roll compaction in rat

目的探讨季铵化壳聚糖-重组组织因子途径抑制物(rTFPI)复合物对大鼠碾压撕脱皮瓣的影响。方法采用实验研究方法。采用离子交联法制备季铵化壳聚糖-rTFPI复合物溶液,用扫描电子显微镜观察复合物形态并测量其直径,用酶联免疫吸附测定(ELISA)法测定并计算复合物中rTFPI的包封率和复合物载药率(样本数为3),采用ELISA法测定放置0、10、30、45、60、90、120、240 min时溶液中rTFPI浓度(观察rTFPI释放情况)并计算其半衰期(样本数为3)。取24只6周龄雄性SD大鼠,按照随机数字表法分为磷酸盐缓冲液(PBS)组、单纯季铵化壳聚糖组、单纯rTFPI组和季铵化壳聚糖-rTFPI组(每组6只),大鼠背部均制备蒂部位于双侧髂棘连线上、从肌膜表面掀起的碾压撕脱皮瓣,原位缝合后即刻及术后1、2、3 d分别注射1次相应试剂治疗。术后1、3、7 d观察皮瓣大体变化;术后7 d,测量皮瓣成活面积并计算皮瓣成活率;将皮瓣平均分为蒂部、近段、中段、远段,用激光散斑血流成像仪检测皮瓣近段、中段、远段组织血流灌注情况;切取皮瓣中段组织行苏木精-伊红染色,观察组织结构变化和炎症细胞浸润情况,计数每100倍视野中栓塞血管和新生血管。对数据行单因素方差分析和LSD检验。结果季铵化壳聚糖-rTFPI复合物呈不规则球形结构,直径为150~200 nm;复合物中rTFPI的包封率为(88.7±2.1)%,复合物的载药率为(2.83±0.09)%。季铵化壳聚糖-rTFPI复合物溶液中rTFPI浓度随放置时间延长而逐渐增加,90 min时释放基本稳定,半衰期为(651±36)min。术后1 d,单纯季铵化壳聚糖组大鼠皮瓣远段变黑比较明显;术后3 d,单纯rTFPI组和季铵化壳聚糖-rTFPI组大鼠皮瓣远段结痂、坏死较另外2组轻;术后7 d,各组大鼠皮瓣坏死界限清晰。术后7 d,单纯rTFPI组和季铵化壳聚糖-rTFPI组大鼠皮瓣成活率分别为(63±7)%、(73±5)%,较PBS组的(41±3)%和单纯季铵化壳聚糖组的(52±7)%显著升高(P<0.05);季铵化壳聚糖-rTFPI组大鼠皮瓣成活率较单纯rTFPI组明显升高(P<0.05)。术后7 d,各组大鼠皮瓣均有血流灌注;单纯季铵化壳聚糖组大鼠皮瓣近段组织血流灌注值以及单纯rTFPI组和季铵化壳聚糖-rTFPI组大鼠皮瓣近段、中段、远段组织血流灌注值明显高于PBS组(P<0.05或P<0.01),单纯rTFPI组大鼠皮瓣远段组织血流灌注值以及季铵化壳聚糖-rTFPI组大鼠皮瓣中段和远段组织血流灌注值明显高于单纯季铵化壳聚糖组(P<0.05或P<0.01),季铵化壳聚糖-rTFPI组大鼠皮瓣中段组织血流灌注值明显高于单纯rTFPI组(P<0.01)。术后7 d,PBS组和单纯季铵化壳聚糖组大鼠皮瓣中段组织中炎症细胞浸润较多,细胞水肿明显;与前面2组相比,单纯rTFPI组和季铵化壳聚糖-rTFPI组大鼠皮瓣中段组织炎症程度显著降低,栓塞血管数明显减少(P<0.05或P<0.01),新生血管数明显增多(P<0.05或P<0.01);与单纯rTFPI组比较,季铵化壳聚糖-rTFPI组大鼠皮瓣中段组织中新生血管数显著增多(P<0.05)。结论通过季铵化壳聚糖装载rTFPI能够起到缓释rTFPI的效果;季铵化壳聚糖-rTFPI复合物较单纯rTFPI能够进一步改善大鼠碾压撕脱皮瓣的血流灌注,提高皮瓣成活率。

Objective To investigate the effect of N-trimethyl chitosan-recombinant tissue factor pathway inhibitor(rTFPI)complex on avulsion flap with roll compaction in rat.Methods The experimental methods were adopted.The N-trimethyl chitosan-rTFPI complex solution was prepared by ion cross-linking method.The morphology of the complex was observed by scanning electron microscope,and its diameter was measured.The encapsulation rate of rTFPI in the complex and drug loading rate of the complex was determined and calculated by enzyme-linked immunosorbent assay(ELISA)method(n=3).The concentration of rTFPI in the solution at 0,10,30,45,60,90,120,240 minutes of storage was measured by ELISA method to observe the release of rTFPI,and its half-life was calculated(n=3).Twenty-four 6-week-old male Sprague-Dawley rats were divided into phosphate buffered saline(PBS)group,N-trimethyl chitosan alone group,rTFPI alone group,and N-trimethyl chitosan-rTFPI group according to the random number table,with 6 rats in each group.The avulsion flaps with roll compaction were prepared on the backs of rats with pedicles located on the line of the bilateral iliac spine and lifted from the surface of the muscle membrane.One injection of corresponding reagents was carried out immediately after in-situ suture and on post operation day(POD)1,2,and 3.General changes of the flap were observed on POD 1,3,and 7.On POD 7,the survival area of the flap was measured and the survival rate of the flap was calculated;the flaps were divided into pedicle,proximal,middle,and distal segments,and the blood perfusion in the proximal,middle,and distal segment tissue of the flap was detected by the laser speckle blood flow imager;tissue samples in the middle of the flap were cut and stained with hematoxylin and eosin to observe the changes in tissue structure and the infiltration of inflammatory cells,and the numbers of embolized blood vessels and new blood vessels per 100 times visual field were counted.Data were statistically analyzed with one-way analysis of variance and least significant difference test.Results The N-trimethyl chitosan-rTFPI complex had an irregular spherical structure with a diameter of 150-200 nm.The encapsulation rate of rTFPI in the complex and drug loading rate of the complex were(88.7±2.1)%and(2.83±0.09)%,respectively.The concentration of rTFPI in the solution of the N-trimethyl chitosan-rTFPI complex gradually increased with prolonged storage time,and the release was basically stable at 90 min,with half-life of(651±36)min.On POD 1,the distal parts of flaps of rats in N-trimethyl chitosan alone group darkened significantly.On POD 3,scabs and necrosis were relatively mild on the distal segment of the flaps of rats in rTFPI alone group and N-trimethyl chitosan-rTFPI group as compared with those of the other two groups.On POD 7,the necrosis boundaries of the flaps of rats in each group were clear.On POD 7,the flap survival rates of rats in rTFPI alone group and N-trimethyl chitosan-rTFPI group were(63±7)%and(73±5)%,respectively,which were significantly higher than(41±3)%in PBS group and(52±7)%in N-trimethyl chitosan alone group.Moreover,the flap survival rate of rats in N-trimethyl chitosan-rTFPI group was significantly higher than that in rTFPI alone group(P<0.05).On POD 7,the flaps of rats in each group had blood perfusion;the blood perfusion values in the proximal segment tissue of the rat flaps in N-trimethyl chitosan alone group and the blood perfusion values in the proximal,middle,and distal segment tissue of the rat flaps in rTFPI alone group and N-trimethyl chitosan-rTFPI group were significantly higher than those in PBS group(P<0.05 or P<0.01);the blood perfusion values in the distal segment tissue of the rat flaps in rTFPI alone group and the blood perfusion values in the middle and distal segment tissue of the rat flaps in N-trimethyl chitosan-rTFPI group were significantly higher than those in N-trimethyl chitosan alone group(P<0.05 or P<0.01);the blood perfusion value in the middle segment tissue of the rat flaps in N-trimethyl chitosan-rTFPI group was significantly higher than that in rTFPI alone group(P<0.01).On POD 7,inflammatory cells infiltrated more and cell edema was obvious in the middle segment tissue of the rat flaps in PBS group and N-trimethyl chitosan alone group.Compared with those of the previous two groups,the inflammation degrees in the middle segment tissue of the rat flaps in rTFPI alone group and N-trimethyl chitosan-rTFPI group were significantly milder,the number of embolized blood vessels was significantly decreased(P<0.05 or P<0.01),and the number of new blood vessels was significantly increased(P<0.05 or P<0.01).Compared with that of rTFPI alone group,the number of new blood vessels in the middle segment tissue of the rat flaps in N-trimethyl chitosan-rTFPI group increased significantly(P<0.05).Conclusions The effect of sustained release of rTFPI can be achieved by loading rTFPI with N-trimethyl chitosan.Compared with rTFPI alone,the N-trimethyl chitosan-rTFPI complex can further improve the blood perfusion of the avulsion flaps with roll compaction in rat and improve the survival rate of the flap.