柠檬酸化半水硫酸钙作为骨移植替代材料的安全性及生物相容性评价

Safety and biocompatibility of citrated calcium sulphate hemihydrate as substitute of bone transplantation

目的:以二水硫酸钙为原料,制备出高纯度柠檬酸化半水硫酸钙,并对其生物相容性进行评价。方法:实验于2004-09/2005-01在解放军总医院骨科研究所完成。柠檬酸化半水硫酸钙粉沫是粉状的二水硫酸钙经特殊处理制得。根据文献对实验材料进行生物相容性及安全性评价实验。①红细胞溶解性:材料粉末制备的浸提液加入抗凝兔血内恒温水浴60min、离心取上清液,分光光度计测定上清液吸光度。计算红细胞溶血率(%)=(实验组吸光度-阴性对照组吸光度)/(阳性对照组吸光度-阴性对照组吸光度)×100%、标准:≤5%为正常。②细胞毒性试验:采用四甲基偶氮唑盐比色法。兔骨髓基质干细胞经复苏、传代、培养24h后进行更换含材料浸提液的培养基继续培养、酶标仪测定1,3,5,7d吸光度。计算细胞相对增值率=实验组吸光度/对照组吸光度。③细胞材料表面贴附:骨髓基质干细胞细胞悬液滴在材料表面,培养3d后,倒置显微镜下观察材料边缘细胞生长情况。乙醇固定后用环境扫描电镜镜下观察材料表面的细胞贴附情况。④热源性:自体温试验筛选合格新西兰家兔的耳缘静脉注入材料浸提液后,定时测兔体温。标准:每只体温升高在0.6℃以下,3只体温升高总度数小于1.4℃。⑤皮肤刺激性:健康新西兰家兔脊柱两侧选点注射材料花生油及DMEM浸提液,每个点注射0.2mL。观察注射部位皮肤反应。⑥致敏性:将浸提液与完全氟氏佐剂完全乳化制成相应剂型试剂,以豚鼠为观察对象,观察腹部激发部位皮肤反应。结果:①材料特性:制备材料2h固化抗压缩强度达到26MPa,初凝时间5min,终凝时间20min。扫描电镜观察为晶体形态均一、为规则的六面体结构。②细胞粘附与生长形态:骨髓基质干细胞在材料周围生长,环境扫描电镜观察细胞贴附材料表面生长,呈不规则的梭形、多角形,胞体伸出伪足长短不等。③红细胞溶解实验:红细胞溶血率为0.34%,远小于阳性标准,说明材料植入体内后不会引起机体本身的溶血反应。④热源反应:家兔平均体温升高为0.1℃,符合医用材料的热源反应要求,说明本材料本身不具有致热源作用。⑤皮内注射:两组浸提液皮内注射后均未引起家兔实验区的红斑及水肿反应,提示材料对动物机体没有刺激性。⑥致敏实验:致敏率为0,与阴性对照没有差别,说明材料没有致敏性。⑦细胞毒性实验的检测结果提示实验组细胞相对增值率和对照组无显著性差别,评分为0～1级,说明材料本身不具有细胞毒性。结论:生物安全性评价结果初步显示制备的材料具有良好的生物相容性,符合医用生物材料的基本要求。

AIM： To prepare citrated calcium sulphate hemihydrate powders materials made from calcium sulphate dihydrate, and evaluate its biocompatability. METHODS： The study was finished in the Institutes of Orthopaedics, General Hospital of Chinese PLA from September 2004 to January 2005. Citrated calcium sulphate powde~ were prepared by processing calcium sulphate dihydrate with special methods. The bioeompatibility and safety of the experimental materials were evaluated according to the references. ① Haemolysis tests： Citrated calcium sulphate hemihydrate extractions were added to prepared diluted rabbit blood with decoagulant. The supernatants were got by centrifugations, and the absorbance was determined with spectrophotometer. Haemolysis rate （%）=（absorbanee in the experimental group-absorbanee in the negative control group）/（absorbance in the positive control group-absorbance in the negative control group）×100%. ≤ 5% was taken as normal. ② Cytotoxicity test： Methyl-thiazol-tetrazolium （MTF） colorimetry was used. After the rabbit mesenehymal cells were recovered, passaged and cultured for 24 hours, the media were changed into media containing materials extractions. The absorbanee was determined with enzyme labeling meter. Relative growth rate= absorbanee in the experimental group/absorbanee in the control group, ③ Cell adherence to materials： Mesenchymal cell suspensions were added to materials surface and incubated for 3 days. Cell adherent growth around materials was observed under inverted phase contrast microscope and cell growth on surface of materials with scanning electron microscope after alcohol fixation. ④ Pyrogenie tests： Material extractions were injected via veins at the edge of ear, and the body temperature of the rabbits was determined for 3 times at fixed times, and the increased degrees were recorded. Standard： the body temperature of each rabbit increased by less than 0.6 ℃, and that of 3 rabhits was increased by less than 1.4 ℃ altogether. ⑤Allergie tests： Materials oil and DMEM extractions were injected subcutaneously into the selected points of bilateral spine of healthy New Zealand rabbits, 0.2 mL for each point, and then evaluated according to the reference standard. ⑥ Skin sensitization test： The extractions and complete Freud＇s adjuvant were completely emulsified to prepare corresponding reagent, and .guinea pigs were used, the abdomen skin and skin responses were observed, and then they were graded according to the standard. RESULTS： ① Property of materials： After 2-hour preparation, the compressive strength of eitrated calcium sulphate hemihydrate solidification was superior to 26 MPa, primary setting time was 5 minutes, and final setting time was 20 minutes. Crystals appearance was similar and appears regular hexahedron structure by observation of scanning electron microscope. ② Cell adherence and growth form： Mesenchymal cells could grow around materials, adhere to and proliferate on the surface of materials. Cell appeared irregular fusiform, polygon and extends numberous long-short and varied-formed protrusions. ③ Haemolysis tests： The rate of haemolysis was 0.34%, which was obviously lower than the positive standard, it was indicated that the in vivo transplantation of the materials could not cause the hemolytic reaction of the body. ④ Pyrogenic reaction： The body temperature of the rabbits averagely increased by 0.1 ~C, which accorded with the demands of medical materials, it was indicated that the material itself had no pyrogenic effect. ⑤ lntradermal injection： No erythema, papule and nodule were observed after intradermal injection inboth groups, indicating that the material has no stimulation to the body. ⑥ Sensitization test： The rate of sensitization was 0, which had no significant difference from negative control, suggesting that the material has no sensitization. ⑦ The results of the cytotoxicity test showed that the relative growth rate was insignificantly different between the experimental group and the control group, the score was 0-1 grade, indicating that the material itself had no toxicity to mesenehymal cell proliferations. CONCLUSION： The results of the evaluation showed that the prepared material had good biocompatibility and accorded with the demands of medical biomaterials.