[Effects of N-trimethyl chitosan-recombinant tissue factor pathway inhibitor complex on avulsion flap with roll...

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.