几种分散剂对纳米羟基磷灰石团聚、细胞内分布和细胞增殖的影响  

Effects of dispersants on aggregation,intracellular distribution and cell proliferation of nanohydroxyapatite

作　　者：王任先[1] 曹晶晶 王宏刚 万奔 刘巍峰[2] Wang Renxian;Cao Jingjing;Wang Honggang;Wan Ben;Liu Weifeng(Beijing Research Institute of Traumatology and Orthopedics,Beijing Jishuitan Hospital,Beijing 100035,China;Department of Orthopedic Oncology Surgery,Beijing Jishuitan Hospital,Beijing 100035,China)

机构地区：[1]北京积水潭医院/北京市创伤骨科研究所,北京市100035 [2]北京积水潭医院骨肿瘤科,北京市100035

出　　处：《中国组织工程研究》2021年第16期2500-2505,共6页Chinese Journal of Tissue Engineering Research

基　　金：北京自然科学基金(7192027),项目负责人:王任先;北京市卫生健康委员会改革试点项目(BMHC-2018-4,BMHC-2019-9,PXM2020_026275_000002),项目参与者:王任先、曹晶晶、王宏刚、万奔。

摘　　要：背景:纳米羟基磷灰石具有优异的生物兼容性、生物活性及可修饰性,但是合成的纳米羟基磷灰石具有高比表面能,使得其在溶液中容易团聚。目的:比较超声以及不同分散剂对纳米羟基磷灰石团聚、细胞毒性及在细胞内分布的影响。方法:采用化学沉淀法合成针状纳米羟基磷灰石,高压灭菌后备用。将不同浓度的分散剂六偏磷酸纳(0,0.25,0.5,1,2 mmol/L)、柠檬酸钠(0,0.25,0.5,1,2 mmol/L)、聚甲基丙烯酸钠(0%,0.0625%,0.125%,0.25%,0.5%)分别与MC3T3E1细胞共培养,采用CCK-8法检测细胞增殖,筛选合适的分散剂作用浓度用于后续实验。将纳米羟基磷灰石溶液分5组处理:对照组不进行分散,其余4组分别进行超声分散、1 mmol/L六偏磷酸纳分散、1 mmol/L柠檬酸钠分散、0.125%聚甲基丙烯酸钠分散,检测纳米羟基磷灰石团聚尺寸。采用超声、1mmol/L六偏磷酸纳、1mmol/L柠檬酸钠、0.125%聚丙烯酸钠分散处理羟基磷灰石后分别与MC3T3E1细胞共培养,以未分散纳米羟基磷灰石与MC3T3E1细胞共培养为对照,采用CCK-8法检测细胞增殖,培养1 d时透射电镜观察下纳米羟基磷灰石在细胞内的分布。结果与结论:①1 mmol/L及更低浓度的六偏磷酸纳无明显的细胞毒性,0.25-2 mmol/L的柠檬酸钠均无明显的细胞毒性,不同浓度的聚甲基丙烯酸钠具有一定的细胞毒性,呈时间与浓度依赖性,后续实验选择1 mmol/L六偏磷酸纳、1 mmol/L柠檬酸钠、0.125%聚丙烯酸钠进行分散处理;②3种分散剂都显著降低了纳米羟基磷灰石的团聚尺寸,其中六偏磷酸纳的分散效果最好;③分散方式和分散剂的加入会显著影响纳米羟基磷灰石的生物学功能,柠檬酸钠会促进与纳米羟基磷灰石共培养的细胞增殖,超声和聚甲基丙烯酸钠会抑制与纳米羟基磷灰石共培养的细胞增殖;④透射电镜显示,对照组和超声组可见较大的纳米羟基磷灰石团聚块,团聚块存在于细�BACKGROUND:Nano-hydroxyapatite has excellent biocompatibility,biological activity,and modifiability,but the synthesized nano-hydroxyapatite has a high specific surface energy,which makes it agglomerate in solution.OBJECTIVE:To compare the effects of ultrasound and different dispersants on the aggregation,cytotoxicity and intracellular distribution of nanohydroxyapatite.METHODS:Needle like nano-hydroxyapatite was prepared by chemical precipitation method,and kept for further use after high pressure sterilization.Different concentrations of dispersant sodium hexametaphosphate(0,0.25,0.5,1,2 mmol/L),sodium citrate(0,0.25,0.5,1,2 mmol/L),sodium polymethacrylate(0%,0.0625%,0.125%,0.25%,0.5%)were co-cultured with MC3 T3 E1 cells.Cell proliferation was detected by CCK-8 assay.The appropriate concentration of dispersant was screened for subsequent experiments.The nano-hydroxyapatite solution was divided into five groups:the control group was not dispersed;and the remaining four groups were subjected to ultrasonic dispersant,1 mmol/L sodium hexametaphosphate dispersant,1 mmol/L sodium citrate dispersant,and 0.125%sodium polymethacrylate dispersant.The aggregate size of nano-hydroxyapatite was detected.The hydroxyapatite was treated with ultrasound,1 mmol/L sodium hexametaphosphate,1 mmol/L sodium citrate,and 0.125%sodium polyacrylate dispersants and then co-cultured with MC3 T3 E1 cells.Undispersed nano-hydroxyapatite co-cultured with MC3 T3 E1 cells was considered as control.Cell proliferation was detected by CCK-8 assay.At 1 day after culture,the distribution of nano-hydroxyapatite was observed by transmission electron microscope.RESULTS AND CONCLUSION:(1)Sodium hexametaphosphate at a concentration of 1 mmol/L and lower had no obvious cytotoxicity.Sodium citrate at 0.25-2 mmol/L had no obvious cytotoxicity.Sodium polymethacrylate at different concentrations had certain cytotoxicity in time-and concentration-dependent manners.Subsequent experiments selected 1 mmol/L sodium hexametaphosphate,1 mmol/L sodium citrate,and 0.

关 键 词：骨 材料 纳米羟基磷灰石 团聚 分散 分散剂 超声 骨移植材料 细胞毒性 细胞分布 细胞增殖 

分 类 号：R459.9[医药卫生—治疗学] R318.08[医药卫生—临床医学]