Effects of N-trimethyl chitosan-recombinant tissue factor pathway inhibitor complex on avulsion flap with roll compaction in rat
-
摘要:
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。 -
关键词:
- 皮肤 /
- 壳聚糖 /
- 血液灌注 /
- 重组组织因子途径抑制物 /
- 碾压撕脱皮瓣
Abstract: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. -
参考文献
(36) [1] McGowanSP, FallahiAKM. Degloving injuries[M]. Treasure Island (FL): StatPearls Publishing, 2020. [2] Boettcher-HaberzethS,SchiestlC.Management of avulsion injuries[J].Eur J Pediatr Surg,2013,23(5):359-364.DOI: 10.1055/s-0033-1353493. [3] 郭杰 皮肤撕脱伤撕脱皮瓣坏死机理的研究 西安 第四军医大学 1997 郭杰.皮肤撕脱伤撕脱皮瓣坏死机理的研究[D].西安:第四军医大学,1997.
[4] EkstrandJ,RazuvaevA,FolkersenL,et al.Tissue factor pathway inhibitor-2 is induced by fluid shear stress in vascular smooth muscle cells and affects cell proliferation and survival[J].J Vasc Surg,2010,52(1):167-175.DOI: 10.1016/j.jvs.2010.02.282. [5] 张旭东,吴近芳,茅东升,等.rTFPI在大鼠预防碾压撕脱皮瓣坏死中的应用[J].中华显微外科杂志,2016,39(4):359-362.DOI: 10.3760/cma.j.issn.1001-2036.2016.04.013. [6] 余波,罗师平,宋丽萍,等.氨基酸序列突变对TFPI生物半衰期和生物活性的影响[J].生物化学与生物物理进展,2007,34(4):382-388.DOI: 10.3321/j.issn:1000-3282.2007.04.008. [7] BaiH,MaD,ZhangYG,et al.Molecular design and characterization of recombinant long half-life mutants of human tissue factor pathway inhibitor[J].Thromb Haemost,2005,93(6):1055-1060.DOI: 10.1160/TH04-11-0721. [8] KravanjaG,PrimožičM,KnezŽ,et al.Chitosan-based (Nano)materials for Novel Biomedical Applications[J].Molecules,2019,24(10):1960.DOI: 10.3390/molecules24101960. [9] ShariatiniaZ,JalaliAM.Chitosan-based hydrogels: preparation, properties and applications[J].Int J Biol Macromol,2018,115:194-220.DOI: 10.1016/j.ijbiomac.2018.04.034. [10] RizeqBR,YounesNN,RasoolK,et al.Synthesis, bioapplications, and toxicity evaluation of chitosan-based nanoparticles[J].Int J Mol Sci,2019,20(22):5776.DOI: 10.3390/ijms20225776. [11] DaiT,TanakaM,HuangYY,et al.Chitosan preparations for wounds and burns: antimicrobial and wound-healing effects[J].Expert Rev Anti Infect Ther,2011,9(7):857-879.DOI: 10.1586/eri.11.59. [12] HuY,ZhanC,ZhouA,et al.Synthesis and characterization of L-tyrosine-conjugated quaternary ammonium salt chitosan and their cytocompatibility as a potential tissue engineering scaffold[J].J Biomater Sci Polym Ed,2020,31(7):833-848.DOI: 10.1080/09205063.2020.1712174. [13] YiY,FeiJ,XuYX,et al.Preparation and characterization of a Schiff-based chitosan-fructose quaternary ammonium salt for medical applications[J].J Biomater Sci Polym Ed,2020,31(6):804-815.DOI: 10.1080/09205063.2020.1719684. [14] BognackiJ,HammelburgerJ.Functional and immunologic methods for the measurement of human tissue factor pathway inhibitor[J].Blood Coagul Fibrinolysis,1995,6 Suppl 1:S65-72.DOI: 10.1097/00001721-199506001-00011. [15] 茅东升,张旭东,郑丽君,等.大鼠碾压撕脱皮瓣模型建立及皮瓣血运情况检测[J].中华烧伤杂志,2015,31(1):58-61.DOI: 10.3760/cma.j.issn.1009-2587.2015.01.015. [16] WangJ,TuoZ,ZhangJ,et al.Hyperoxygenated solution improves tissue viability in an avulsion injury flap model[J].J Plast Reconstr Aesthet Surg,2020,73(5):975-982.DOI: 10.1016/j.bjps.2019.11.029. [17] 金文虎,周爱婷,吴中桓,等.血管内皮生长因子基因修饰人羊膜间充质干细胞利于超长随意皮瓣的成活[J].中国组织工程研究,2019,23(21):3349-3356.DOI: 10.3969/j.issn.2095-4344.1743. [18] XuP,WuY,ZhouL,et al.Platelet-rich plasma accelerates skin wound healing by promoting re-epithelialization[J/OL].Burns Trauma,2020,8:tkaa028[2020-09-14].https://pubmed.ncbi.nlm.nih.gov/32821743/.DOI: 10.1093/burnst/tkaa028. [19] 李璐含,张秋菊,仉红刚,等.激光多普勒血流探测仪小波分析早期高血压大鼠血流灌注频谱特征[J].微循环学杂志,2019,29(2):1-7,13.DOI: 10.3969/j.issn.1005-1740.2019.02.001. [20] 刘晓庆,仉红刚,张秋菊,等.高功率多普勒血流探测仪结合血流成像仪观察氯吡格雷对高血压大鼠微循环的影响?[J].微循环学杂志,2015,25(2):30-33.DOI: 10.3969/j.issn.1005-1740.2015.02.009. [21] SamsiS,JonesM,KepnerJ,et al.Colorization of H&E stained tissue using Deep Learning[J].Annu Int Conf IEEE Eng Med Biol Soc,2018,2018:640-643.DOI: 10.1109/EMBC.2018.8512419. [22] PowersDB,BreezeJ.Avulsive soft tissue injuries[J].Atlas Oral Maxillofac Surg Clin North Am,2019,27(2):135-142.DOI: 10.1016/j.cxom.2019.05.005. [23] 宋坤修,马丙栋,刘永涛.吻合回流静脉对手部皮肤逆行撕脱伤成活的影响[J].实用手外科杂志,2018,32(1):100-101.DOI: 10.3969/j.issn.1671-2722.2018.01.034. [24] 郭杰,鲁开化,郭树忠,等.皮肤撕脱伤撕脱皮瓣组织血管活性因子的变化及意义[J].1997,20(3):221-222.DOI: 10.3760/cma.j.issn.1001-2036.1997.03.131. [25] 宋保强,郭树忠,鲁开化,等.皮肤撕脱伤中CD11b、ICAM-1动态表达的实验研究[J].中国美容医学杂志,2002,11(3):205-207.DOI: 10.3969/j.issn.1008-6455.2002.03.005. [26] LantieriLA,OzbekMR,DeuneEG,et al.Prevention of microvascular thrombosis by topical application of recombinant tissue factor pathway inhibitor[J].Plast Reconstr Surg,1996,97(3):587-594.DOI: 10.1097/00006534-199603000-00015. [27] 吴耀强,陈宁,赖胜华,等.低分子肝素钙不同注射方式治疗带蒂皮瓣静脉危象的效果对比[J].中国当代医药,2018,25(30):48-50.DOI: 10.3969/j.issn.1674-4721.2018.30.016. [28] 韩素琴,王欣,胡浩良,等.改良远端蒂腓动脉穿支螺旋桨皮瓣修复小腿及足踝部软组织缺损的围手术期护理[J].中华显微外科杂志,2018,41(2):201-202.DOI: 10.3760/cma.j.issn.1001-2036.2018.02.030. [29] 荆文慧,荆洁.低分子量肝素钙所致不良反应文献概述[J].中国药物滥用防治杂志,2020,26(2):123-124.DOI: 10.15900/j.cnki.zylf1995.2020.02.013. [30] KhouriRK,ShermanR,BunckeHJ,et al.A phase Ⅱ trial of intraluminal irrigation with recombinant human tissue factor pathway inhibitor to prevent thrombosis in free flap surgery[J].Plast Reconstr Surg,2001,107(2):408-415; discussion 416-418.DOI: 10.1097/00006534-200102000-00016. [31] ChowdaryP.Anti-tissue factor pathway inhibitor (TFPI) therapy: a novel approach to the treatment of haemophilia[J].Int J Hematol,2020,111(1):42-50.DOI: 10.1007/s12185-018-2548-6. [32] AhnströmJ,GierulaM,TemenuJ,et al.Partial rescue of naturally occurring active site factor X variants through decreased inhibition by tissue factor pathway inhibitor and antithrombin[J].J Thromb Haemost,2020,18(1):136-150.DOI: 10.1111/jth.14627. [33] MaroneySA,HansenKG,MastAE.Cellular expression and biological activities of alternatively spliced forms of tissue factor pathway inhibitor[J].Curr Opin Hematol,2013,20(5):403-409.DOI: 10.1097/MOH.0b013e3283634412. [34] SantamariaS,Reglińska-MatveyevN,GierulaM,et al.Factor V has an anticoagulant cofactor activity that targets the early phase of coagulation[J].J Biol Chem,2017,292(22):9335-9344.DOI: 10.1074/jbc.M116.769570. [35] YehYC,HuangTH,YangSC,et al.Nano-based drug delivery or targeting to eradicate bacteria for infection mitigation: a review of recent advances[J].Front Chem,2020,8:286.DOI: 10.3389/fchem.2020.00286. [36] SongM,WangH,ChenK,et al.Oral insulin delivery by carboxymethyl-β-cyclodextrin-grafted chitosan nanoparticles for improving diabetic treatment[J].Artif Cells Nanomed Biotechnol,2018,46(Suppl 3):S774-782.DOI: 10.1080/21691401.2018.1511575. -
4 4组大鼠碾压撕脱皮瓣术后各时间点大体情况。4A、4B、4C.分别为磷酸盐缓冲液组术后1、3、7 d皮瓣情况，3 d时皮瓣中远段变黑明显且远段开始结痂坏死；4D、4E、4F.分别为单纯季铵化壳聚糖组术后1、3、7 d皮瓣情况，1 d时皮瓣远段变黑明显，3 d时皮瓣远段开始结痂；4G、4H、4I.分别为单纯重组组织因子途径抑制物（rTFPI）组术后1、3、7 d皮瓣情况，皮瓣远段结痂坏死情况分别较图4A、4B、4C与图4D、4E、4F轻；4J、4K、4L.分别为季铵化壳聚糖-rTFPI组术后1、3、7 d皮瓣情况，皮瓣结痂坏死情况最轻，坏死范围最小
5 4组大鼠碾压撕脱皮瓣术后7 d皮瓣血流灌注情况。5A.磷酸盐缓冲液组皮瓣血流灌注区域相对较少；5B.单纯季铵化壳聚糖组皮瓣血流灌注区域与图5A相近；5C.单纯重组组织因子途径抑制物（rTFPI）组皮瓣血流灌注区域较图5A、5B扩大；5D.季铵化壳聚糖-rTFPI组皮瓣大部分区域有血流灌注
注：图中紫红色表示有血流灌注，蓝色表示血流灌注少，黑色表示无血流灌注
6 4组大鼠碾压撕脱皮瓣术后7 d皮瓣中段组织病理情况 苏木精-伊红×100。6A.磷酸盐缓冲液组细胞水肿明显；6B.单纯季铵化壳聚糖组炎症细胞浸润较多，可见细胞水肿；6C.单纯重组组织因子途径抑制物（rTFPI）组炎症细胞浸润减少，可见新生毛细血管；6D.季铵化壳聚糖-rTFPI组新生毛细血管较图6C增多
注：蓝色箭头标记水肿细胞，黑色箭头标记炎症细胞，红色箭头标记新生血管
表1 4组大鼠碾压撕脱皮瓣术后7 d各部分组织血流灌注情况比较（
组别 鼠数（只） 近段 中段 远段 PBS组 6 138±33 122±11 94±23 单纯季铵化壳聚糖组 6 176±17a 134±36 80±42 单纯rTFPI组 6 182±42a 154±17 b 130±26be 季铵化壳聚糖-rTFPI组 6 189±24b 190±22 bcd 162±44bc F值 3.889 11.432 7.808 P值 <0.05 <0.01 0.01 注：与磷酸盐缓冲液（PBS）组比较，aP<0.05，bP<0.01；与单纯季铵化壳聚糖组比较，cP<0.01，eP<0.05；与单纯重组组织因子途径抑制物（rTFPI）组比较，dP<0.01 
下载: 导出CSV
表2 4组大鼠碾压撕脱皮瓣术后7 d中段组织中每100倍视野下血管情况比较（根，
组别 鼠数（只） 栓塞血管数 新生血管数 PBS组 6 16.2±4.3 7.4±1.7 单纯季铵化壳聚糖组 6 15.6±3.0 6.8±1.9 单纯rTFPI组 6 9.8±3.8ab 10.6±2.1cd 季铵化壳聚糖-rTFPI组 6 7.6±1.1ac 14.0±2.2ace F值 8.248 13.924 P值 0.002 0.000 注：与磷酸盐缓冲液（PBS）组比较，aP<0.01，dP<0.05；与单纯季铵化壳聚糖组比较，bP<0.05，cP<0.01；与单纯重组组织因子途径抑制物（rTFPI）组比较，eP<0.05
下载: 导出CSV
-
图(6) / 表(2)
计量
- 文章访问数: 164
- HTML全文浏览量: 122
- PDF下载量: 21
- 被引次数: 0
