摘要
目的本实验旨在观察中药骨碎补提取物骨碎补总黄酮(AFDR)复合海藻酸基可注射骨修复材料对成骨细胞MC3T3-E1体外培养的影响。方法将成骨细胞株MC3T3-E1随机分为空白对照组、单纯使用海藻酸基可注射骨修复材料组、AFDR0.04mg/L、0.16mg/L、0.64mg/L、1.28mg/L、5.12mg/L 7组,体外复合海藻酸基可注射骨修复材料,以24h和48h为观察时间点,倒置显微镜观察MC3T3-E1细胞形态,台盼蓝拒染法检测MC3T3-E1存活率,MTT法观察细胞增殖效应,碱性磷酸酶(ALP)活性和骨钙素定量检测分别观察不同浓度骨碎补总黄酮促进MC3T3-E1分化作用,采用Von kossa钙化染色法观察其促MC3T3-E1细胞钙化作用。结果①倒置显微镜下观察复合海藻酸基可注射骨修复材料的MC3T3-E1细胞形态,细胞形态呈三角形、多边形、长梭形等,呈集落样生长,无接触抑制,有伪足伸出,胞液透亮,核圆形。AFDR各组不同程度促进成骨细胞的增殖、分化:7组能不同程度的促进成骨细胞的增殖、分化,与空白对照组比较,以0.16 mg/L和0.64mg/L剂量组的成骨细胞增殖数量最多,1.28mg/L,5.12mg/L的AFDR对细胞增殖作用不明显;②MC3T3-E1平均存活率≥90%;③骨碎补总黄酮(AFDR)0.16 mg/L和0.64mg/L剂量组在培养24小时和48小时,其OD值与空白对照组及组间均有显著性差异,可促进MC3T3-E1细胞增殖(P<0.05和P<0.01);④AFDR 0.16 mg/L和0.64mg/L剂量组能使MC3T3-E1的ALP活性升高(P<0.05);⑤AFDR 0.64mg/L剂量组能促进MC3T3-E1骨钙素合成(P<0.05);⑥AFDR 0.16 mg/L和0.64mg/L剂量组均可促进细胞钙化(P<0.05)。结论 AFDR可显著促进在海藻酸基可注射骨修复材料上MC3T3-E1的增殖、分化和钙化,其作用具有时间依赖和剂量依赖关系。
Objective This study was designed to observe the effect of AFDR in combination with alginate-based injectable bone repair materials on osteoblast MC3T3-E1 cultured in vitro.Methods MC3T3-E1 cells were randomly divided into control group,pure materials group,AFDR0.04mg/L,0.16mg/L,0.64mg/L,1.28mg/L,5.12mg/L 6 group.The observation time point:24h and 48h,inverted microscope MC3T3-E1 cell morphology,trypan blue staining MC3T3-E1 cell viability.THE effects were observed by MTT cell proliferation.All data are expressed as mean ± standard deviation,using SPSS 13.0 software for analysis of variance.The effect of different concentrations of Total Flavonoids promoting differentiation of MC3T3-E1 was observed by ALP activity and osteocalcin quantitative detection.The effect of promoting calcification of MC3T3-E1 cells were observed by Von kossa calcification staining.Results ①inverted microscope composite alginate-based injectable bone repair materials MC3T3-E1 cell morphology,cell morphology triangular,polygonal,fusiform,etc,were colony-like growth,no contact inhibition,extending pseudopodia,cytosol translucent,round nuclei.Different sets of degrees AFDR promote osteoblast proliferation and differentiation;②MC3T3-E1 average survival rate≥90%;③ AFDR 0.64mg / L dose group was cultured for 24h and 48h,its OD value with the blank control group and the groups were significantly different,with statistical significance(P<0.05 and P<0.01);④ AFDR 0.16 mg / L and 0.64mg/L dose can increase activity of ALP of MC3T3-E1(P<0.05);⑤ AFDR 0.64mg/ml dose can promote the synthesis of osteocalcin of MC3T3-E1(P<0.05);⑥AFDR 0.16 mg / L and 0.64mg/L dose groups can promote cell calcification(P<0.05).Conclusion AFDR can significantly facilitate the alginate-based injectable bone repair material on the proliferation、differentiation and calcificationof MC3T3-E1,its role has a time-dependent and dose-dependent manner.
出处
《生物骨科材料与临床研究》
CAS
2014年第2期53-57,74,共6页
Orthopaedic Biomechanics Materials and Clinical Study
基金
教育部新世纪优秀人才支持计划基金项目(NCET-10-0249)
北京中医药大学青年教师资助项目
北京中医药大学青年基金(2012-QNJSZX-016)
