脱细胞血管基质与血管平滑肌细胞的体外相容性

Compatibility of acelluarized allogenic matrix and smooth muscle cells in vitro

目的:酶-化学除垢剂制备脱细胞血管基质,检测其与平滑肌细胞的相容性,探讨其作为组织工程支架材料的可行性。方法:实验于2003-12/2005-12在哈尔滨医科大学附属第二医院动物实验中心完成。①脱细胞血管基质制备:在37℃水浴振荡条件下,犬总动脉先后用1g/L胰蛋白酶+0.2g/L乙二胺四乙酸钠和1.0%TritonX-100进行脱细胞处理24h和176h,充分漂洗,冷冻干燥后备用。②平滑肌细胞的提取:应用组织块贴壁法体外培养扩增犬隐静脉的平滑肌细胞。③四甲基偶氮唑盐法测定脱细胞血管基质浸提液对平滑肌细胞的毒性:设浸提原液、75%、50%和25%浸提液组,阴性对照组(DMEM+体积分数为0.1的胎牛血清培养液)。以阴性对照组的吸光度作为100%细胞增殖率,增殖百分率(P%)=犤实验组A值/阴性对照组A值犦×100%,按ISO和医学生物材料标准评定材料毒性。④急性毒性试验:取成年大鼠8只,将备用脱细胞血管基质研磨成粉末,生理盐水配成2g/L的混悬液,无菌条件下向大鼠腹腔内注入1mL脱细胞血管基质混悬液,观察72h,记录动物有无死亡及不良反应。⑤亚急性毒性试验:取成年大鼠12只,随机分成2组。实验组用脱细胞血管基质混悬液灌胃,剂量为500mg/kg质量,隔日1次,共10次;对照组给等量生理盐水灌胃。实验期间观察记录体质量变化及动物活动情况。21d取血检查血常规。⑥溶血试验:取抗凝兔血8mL,加入生理盐水10mL,稀释备用。取脱细胞血管基质粉末,加生理盐水中,再加入稀释兔血离心后取上清测定545nm处的吸光度。阴性对照为生理盐水10mL加入稀释兔血;阳性对照为蒸馏水加入稀释兔血。溶血率=犤(试验材料的吸光度-阴性对照的吸光度)/(阳性对照的吸光度-阴性对照的吸光度)犦×100%。⑦凝血试验:血库健康献血员新鲜血浆,用自动血液凝固测定仪进行凝血酶原凝固试验、测定部分凝血活酶凝固时间、凝血酶凝固时间和纤维蛋白原。取血浆2mL加入脱细胞血管基质粉100mg,混合后再测定上述指标。结果:①酶-化学除垢剂能使犬总动脉细胞几乎完全脱落,使基质纤维的三维结构变得疏松。苏木精-伊红、胶原染色和弹力纤维染色显示脱细胞血管基质无细胞残留,主要成分为胶原和弹力纤维。②动物实验表明材料无急性、亚急性毒性。体外实验脱细胞血管基质的溶血率为1.6%,符合医用生物材料生物性能测试所提出的溶血率<5%标准。材料混合前后,血浆中凝血酶原凝固试验、部分凝血活酶时间、凝血酶凝固时间和纤维蛋白原4项指标检测结果无明显变化,说明材料不引起凝血功能的改变。③四甲基偶氮唑盐显示75%脱细胞血管基质浸提液培养平滑肌细胞1,3和5d后,细胞增殖率均大于95%,材料毒性为0或1级。结论:TritonX-100和胰蛋白酶制备的脱细胞基质具有良好的生物相容性,能与异体犬平滑肌细胞结合到一起,并在体外生成血管移植物。

AIM： To prepare acelluarized allogenic matrix with enzyme-chemical scour, and detect its the compatibility with smooth muscle cells （SMCs）, and probe into the feasibility of using acelluarized allogenic matrix as scaffold in tissue engineering. METHODS： The experiment was performed in the Experimental Center of Second Hospital Affiliated to Harbin Medical University from December 2003 to December 2005. ①Preparation of acelluarized allogenic matrix： Under the shaking in water at 37 ℃, the common vessels of dogs were completely rinsed with 1 g/L trypsin and 0.2 g/L ethylenediamine tetraacetic acid （EDTA） solution for 24 hours as well as 1.0 % Triton X-100 for 176 hours consecutively, and then freeze-dried for using.②Obtaining of SMCs： method of tissue culture was adopted to culture in vitro and isolate saphenous SMCs from expanded alloaorta.③The cell-toxicity of acelluarized allogenic matrix to SMCs were detected by means of MTT. The leaching liquor was divided into 75% leaching liquor group, 50% leaching liquor group, 25% leaching liquor group, positive control group [DMEM ＋10% fetal bovine serum （FCS）]. The absorbance of negative control group was taken as 100% cell proliferation rate. Percentage of proliferation （P%） = [A value of experimental/A value of the negative control group] ×100%. The toxicity of scaffold was evaluated according to ISO and criteria for medical biomaterials.④Acute toxicity test： Eight adult rats were selected. The acelluarized allogenic matrix was grinded into powder and made into 2 g/L suspension by dissolving into normal saline, 1 mL of which was injected into the abdomen of rats under aseptic condition to observe for 72 hours. The death and side effects in animals were recorded. ⑤ Subacute toxicity test： 12 adult rats were selected and randomly divided 2 groups. Rats in the experimental group were given intragastric administration of suspension at the dose of 500 mg/kg once every other day for totally 10 days. Rats in thecontrol group were given intragastric administration of normal saline at the same volume. The changes of body mass and action of rats in the course of study were observed and recorded, and blood were routinely detected on the 21^st day.⑥Hemolysis test： 8 mL of anticoagulation cony blood was diluted by 10 mL of normal saline, which was then added into the suspension for centrifugation, and the absorbance of supematant was determined at 545 nm. The diluted cony blood o added with 10 mL of normal saline was taken as the negative control group, while the diluted cony blood added with distilled water was taken as the positive control group. Hemolytic rate = [（absorbance of test material - absorbance of negative control）/（absorbance of positive control - absorbance of negative control） ×100%. ⑦Thrombotest： fresh plasma obtained from healthy donors were determined in the plasmozyme coagulation test by automatic coagulation radiometer. Partial thromboplastin clotting time, thromboplastin clotting time and the content of fibrin were measured. 2 mL of serum was mixed with 100 mg of acelluarized allogenic matrix, and the indicators mentioned-above were determined again. RESULTS：①Enzyme-chemical scour could lead to complete shedding off of common artery cells in dogs and loosen the 3-dimensional structure of matrix fiber. There were no cells on the acelluarized allogenic matrix by HE, collagen dyeing and elastic fibers dyeing, and it was mainly composed of collagen and elastic fibers. ②The animal experiment showed that there were on acute and subacute toxicity on the acelluarized allogenic matrix. In vitro experiment suggested that the hemolytic rate of acellularized matrix was 1.6%, which was in accordance with the criteria of 〈 5% in the biolog ical function test of biomaterial. There were no changes in plasmozyme coagulation test, part thromboplastin clotting time, thromboplastin clotting time and the content of fibrin before and after the experiment, which indicated that the materials could not change the coagulation function. ③MTT showed that the proliferation rate of SMCs was greater than 95% respectively at 1, 3 and 5 days after culture with 75% of acellularized allogenic matrix, and the material toxicity was of zero or grade Ⅰ. CONCLUSION： Acellular matrix produced by Triton X-100 and trypsin has satisfactory biocompatibility to combine with allogenic SMCs, which can generate vasotransplantation in vitro.