改性羟基磷灰石骨修复纳米复合材料的制备及生物学评价

Preparation and biological evaluation of the nano-composite of modified hydroxyapatite as a bone repairing material

目的:制备羟基磷灰石/聚乳酸聚乙醇酸骨修复材料,并对其进行生物学评价。方法:实验于2006-06/2007-02在中科院长春应用化学研究所完成材料制备,在吉林大学基础医学院实验动物中心完成动物实验。将低聚乳酸的羧基与羟基磷灰石表面的钙原子用化学键连接,得到表面接枝聚左旋乳酸的羟基磷灰石,将其与聚乳酸聚乙醇酸共混,得到复合材料PLLA-g-HA/PLGA。溶于氯仿后铺膜(厚0.2mm),用DMEM培养液浸泡材料膜制备浸提液。首先,进行材料生物安全性实验:①细胞毒性实验:将浸提液与培养液混合,接种兔成骨细胞,培养24h,MTT法检测细胞增殖,计算细胞增殖率和细胞毒性级(细胞毒性级0或1级为合格)。②全身毒性实验:小鼠以50mL/kg的剂量静脉注射浸提液,观察72h内小鼠中毒症状。③皮肤刺激实验:兔脊柱两侧皮内注射材料浸提液,观察72h内皮肤有无异常反应。④热原实验:自兔耳缘静脉注入浸提液(10mL/kg)。注射后每0.5h测肛温1次,共6次,以6次中最高的1次减去正常体温,计为升高度数。其次,对复合材料进行细胞黏附性检测:将复合材料制成1%氯仿溶液,涂于硅化的盖玻片上,置于6孔板,每孔接种1×105个成骨细胞,培养3d,在2,24,72h行FITC荧光染色,数码摄像系统拍摄细胞荧光照片。结果:制备了新型PLLA-g-HA/PLGA复合材料。①生物安全性实验结果:MTT实验检测复合材料细胞增殖率为94.8%,细胞毒性级为1级;全身毒性实验中动物无死亡、惊厥、瘫痪、呼吸抑制、腹泻和体质量下降等不良反应;热原实验中兔体温最大的变化值是0.25℃(国家标准为<0.6℃);皮肤刺激实验中未见任何刺激反应,无红斑、焦痂、水肿表现。②细胞黏附性实验结果:细胞接种后2h可见少量细胞开始贴壁;24h时可见贴壁细胞明显增多,并呈聚集生长;培养3d后可见细胞逐渐融合,细胞状态良好。结论:新型PLLA-g-HA/PLGA复合材料符合生物材料细胞毒性要求,按毒性剂量分级属无毒级,无致热原性、对皮肤无刺激作用,具有良好的生物相容性和细胞黏附性。

AIM： To prepare the composite of poly（lactic-co-glycolic acid） （PLGA） and hydroxyapatite （HA） nano-particles surface modified with poly（L-lactic acid） （PLLA） for bone repairing, and evaluate the biocompatibUity of the material. METHODS： The materials were prepared in Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, and the animal tests were fulfilled in Animal Experimental Center of Basic Medical College of JUin University from June 2006 to February 2007. The HA particles were directly grafted with L-lactic acid oligomer that was reacted with the calcium ion on the surface of HA particles to form a calcium carboxylate bond, and the HA particles grafted with PLLA were obtained. Then, PLLA-g-HA and PLGA were blended together to get the copolymer. The composite was dissolved in chloroform solution to make the film with 0.2 mm thickness. Then the extracts of the material in DMEM for 24 hours were obtained. Firstly, biological safety detection of the materials was performed： ①cytotoxicity test： The extracts were mixed with DMEM to seed rabbit osteoblasts for 24 hours. The call proliferation was detected by MTT method. The call proliferation rate and toxicity grade were also calculated （cytotoxicity of Grade 0 or 1 was qualified）.②generel toxicity test： The extracts were injected into mouse vein with the dose of 50 mL/kg. The toxic symptoms were observed within 72 hours.③skin stimulation test： The extracts were injected into the bilateral sides of rabbit spine subcutaneously. The skin exceptional reaction was observed within 72 hours. ④pyrogenic test： The extracts were injected into the rabbit ear vein with the dose of 10 mL/kg. The anus temperature was measured per 0.5 hour for six times. The elevatory degree was calculated by subtracting the natural temperature from the highest one. Secondly, cellular adhesion test of the material composite was performed. The composite was dissolved in chloroform to 1% polymer solution. Then, it was casted on the cover glasses treated with dimethyl dichlorosilane. The samples were put in 6-well tissue culture plates, totally 1 ×10^5 osteoblasts were seeded each well and cultured for 3 days. The cells were dyed with FITC at 2, 24 and 72 hours of culture, and fluorescence photography was performed using digital video system. RESULTS, The novel copolymer of PLLA-g-HNPLGA was prepared. ① Biological safety： The cell viability of the composite was 94.8% and cytotoxicity was Grade 1. The general toxicity test showed that there were no adverse effects in the animals, such as death, convulsion, paralysis, respiratory depression, diarrhea and decreasing weight; The maximal variety of body temperature in rabbits were 0.25 %, which accorded with the national standard （〈 0.6 %）. No erythema, eschar or edema appeared in the skin stimulation test.②Cell adhesion： A few cells began to adhere on the cover glasses after inoculation for 2 hours, adhesive calls manifolded obviously and assembled 24 hours later, inosculated gradually 3 days later, and the cells were in good fettle. CONCLUSION： The nano-composlte of PLLA-g-HNPLGA accords with cytotoxicity demand of the biological materials, presenting no toxicity, pyrogenicity or skin stimulation. It possesses good biocompatibility and cellular adhesion.