原位生长纳米羟基磷灰石晶须增强β-磷酸三钙多孔支架的生物安全性

Biological security of nano-hydroxyapatite wisker and β-tricalcium phosphate composited bone substitute

背景:通过纳米羟基磷灰石原位生长明显提高了磷酸钙支架的强度与韧性。目的:体外评价纳米羟基磷灰石晶须/β-磷酸三钙(nHAW/β-TCP)作为人工骨支架材料的生物相容性。方法:急性全身毒性试验:30只小白鼠随机分为静脉实验组,腹腔实验组和对照组,分别注射浸提液及生理盐水,24,472h观察动物的一般状态。溶血试验:材料浸提液与稀释人鲜血混合观察红细胞溶解情况,545nm下检测A值计算溶率;致敏试验:16只豚鼠随机分为实验组、阴性对照组和阳性对照组,每只豚鼠脊柱两侧皮内注射等体积nHAW/β-TCP架材料浸提液、生理盐水及二硝基氟苯。于注射后即刻和24,48,72h观察局部皮肤反应。细胞毒性试验:材料浸提液养细胞进行细胞形态大体观察,采用CCK-8法观察细胞活性。结果与结论:急性全身毒性试验:人工骨浸提液静脉及腹腔注射后不引起小鼠呼吸、进食改变或死亡,体质量稳定。溶试验:nHAW/β-TCP的溶血率小于ISO规定的5%,可认为这种材料无溶血作用。致敏试验:豚鼠皮内注射后未出现过反应。细胞毒性试验:CCK-8细胞毒性试验显示不同浓度人工骨浸提液的细胞毒性为0级。提示nHAW/β-TCP复合支架引起全身毒性反应、溶血反应和过敏反应,且无细胞毒性,生物相容性良好,符合组织工程人工骨支架材料的应用要求。

BACKGROUND：The technique,which reinforces porous calcium phosphate scaffold by nano hydroxylapatite whisker in situ,can enhance the strength and toughness of scaffold.OBJECTIVE：To evaluate the biocompatibility of the nano-hydroxyapatite wisker and β-tricalcium phosphate（nHAW/β-TCP） as tissue engineered vascular scaffold in vitro. METHODS：The acute systemic toxicity test：Totally 30 mice were divided into intravenous injection group,intra-abdominal injection group and control group.Mice in the three groups were injected by leaching liquor and saline respectively,then the general state of the mice was recorded at the 24,48 and 72 hours.Test of hemolysis：Dissolution of red blood cells were observed in the mixed liquids of leaching liquor and diluted blood,and the A value was detected at optical density 545 nm then the hemolytic rare was calculated.The sensitization test：Sixteen guinea pigs were randomly divided into experimental group,negative control group and postive control group.Leaching liquor from nHAW/β-TCP,normal saline and 2% dinitrofluorobenzene were injected intradermally into each side of the backbone of each guinea pig.The erythema and edema were recorded at immediate,24,48,72 hours.Test of cytotoxicity：The cells were cultured in the special L-DMEM that nHAW/β-TCP were dipped in and then general morphology of cells and cell activity were observed through microscope and CCK-8.RESULTS AND CONCLUSION：The acute systemic toxicity test：After intravenous and intra-abdominal injection of soaked of materal,there were no changes in breath,appetite,body weight of mice and no death.Test of hemolysis：The haemolytic rates of nHAW/β-TCP were less than 5% of international standard organization requirements,suggesting these materaials had no haematolysis effect.The sensitization test：No allergic reaction were found in guinea pig after endodermal injection.Test of cytotoxicity：Test of CCK-8 colormetry showed that the different densities leaching liquor had no cytotoxicity.nHAW/β-TCP does not cause acute whole body toxic reaction,haematolysis effect,allergic reaction and no cytotoxity.The materials have a good biocompatibility and meet the application requirements for tissue engineered vascular scaffold.