不同浓度硝酸银负载珊瑚羟基磷灰石的细胞毒性实验

Cytotoxicity of coral hydroxyapatite coated by silver nitrate at different concentrations

背景:硝酸银负载珊瑚羟基磷灰石是一种具有抗菌性能的新型植骨材料,在国外已有大量的研究报道,但国内对其研究仅处于起步阶段。目的:制备载银珊瑚羟基磷灰石人工骨材料并观察其对L929细胞的毒性特征。设计、时间及地点:细胞学体外对比观察实验,于2008-10/11在解放军广州军区广州总医院中心实验室完成。材料:将自制的珊瑚羟基磷灰石浸泡于1×10-2,1×10-3,5×10-4,1×10-4,5×10-5,1×10-5mol/L硝酸银溶液中制得不同浓度的载银人工骨材料。按1.25cm2材料表面积/1mL培养基比例加入培养基,37℃下浸提24h。方法:将材料浸提培养基与L929细胞共培养48h,运用MTT法测得细胞相对增殖度,用倒置相差显微镜观察细胞形态,结合中华人民共和国国家标准:GB/T16175-2008中的相关标准进行分析。主要观察指标:L929细胞在不同材料上的增殖情况及细胞的形态学变化。结果:MTT法检测结果表明硝酸银浓度≤5×10-5mol/L时,材料对L929细胞生长无明显抑制作用;材料浸提液即使在高浸提比100%下,浸提产物也无细胞毒性作用。镜下观察发现5×10-5mol/L组及1×10-5mol/L组细胞形态正常,贴壁生长良好。结论:低浓度的载银珊瑚羟基磷灰石对体外培养的L929细胞的增殖、生长无明显毒性作用。

BACKGROUND： Coral hydroxyapatite coated by silver nitrate is a new bone graft material, characterizing by good antisepticise, which is initially reported in China. OBJECTIVE： To make silver nitrate-coated coral hydroxyapatite bone substitute and observe the effect on cytotoxicity of L929 cells. DESIGN, TIME AND SETTING： An in vitro cytological contrast study was performed at Central Laboratory of Guangzhou General Hospital of Guangzhou Military Command of Chinese PLA from October to November 2008. MATERIALS： Coral hydroxyapatite was dipped in silver nitrate at different concentrations of 1×10-2, 1×10-3, 5×10-4, 1×10-4, 5×10-5, and 1×10-5 mol/L. The obtained silver nitrate-coated coral hydroxyapatite was incubated in culture media according to the ratio of surface area （1.25 cm2） and culture media （1 mL） at 37 ℃ for 24 hours. METHODS： Silver nitrate-coated coral hydroxyapatite was co-cultured with L929 cells for 48 hours. MTT assay was used to measure relative growth rate; inverted microscope was used to observe cell morphology; the results were analyzed based on national standard of GB/T16175-2008. MAIN OUTCOME MEASURES： Proliferation and morphology of L929 cells on different materials. RESULTS： MTT assay indicated that when concentration of silver nitrate was ≤5×10-5 mol/L, materials had no inhibitory effect on L929 cell growth. The extract productions of the bone substitute did not show cytotoxic effect even at 100%. The cell morphology was normal in the 5×10-5 mol/L and 1×10-5 mol/L groups, and cells were adherent to the wall. CONCLUSION： Silver nitrate-coated coral hydroxyapatite at a low concentration has no toxic effect on proliferation and growth of L929 cells.