壳聚糖/胡须/磷酸钙骨水泥复合生物材料的力学性能及对诱导多能干细胞成骨潜能的影响  

Mechanical properties and effect on osteodifferentiation of induced pluripotent stem cells of chitosan/whisker/calcium phosphate cement composite biomaterial

作　　者：康明[1] 黄杰华[1] 张理选[1] 王新光[1] 郭汉明[1] 何瑞轩 KANG Ming;HUANG Jiehua;ZHANG Lixuan;WANG Xinguang;GUO Hanming;HE Ruixuan(Department of Joint Surgery, Huizhou Central People＇s Hospital, Huizhou Guangdong, 516001, P.R.China)

机构地区：[1]惠州市中心人民医院关节外科,广东惠州516001

出　　处：《中国修复重建外科杂志》2018年第7期959-967,共9页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(31328008);广东省自然科学基金资助项目(2014A030313275)~~

摘　　要：目的观察新型强化型磷酸钙骨水泥(calcium phosphate cement,CPC)生物支架材料的力学性能以及对诱导多能干细胞(induced potential stem cells,i PS)生物活性及成骨潜能的影响。方法根据制备的支架材料不同,将实验分为4组,A组为单纯CPC支架组,B组为CPC∶10%wt壳聚糖为2∶1比例混合支架组,C组为CPC∶10%wt壳聚糖∶胡须为2∶1∶1比例混合支架组,D组为CPC∶10%wt壳聚糖∶胡须为2∶1∶2比例混合支架组。检测各组支架材料力学性能(抗弯强度、断裂功、硬度及弹性模量)。与第5代i PS-MSCs复合培养1、3、7、14 d采用细胞计数试剂盒8(cell counting kit 8,CCK-8)法检测各组吸光度(A)值;1、7、14 d行ALP活性检测、Live/Dead荧光染色及定量检测、RT-PCR检测成骨基因ALP、Runx2、Ⅰ型胶原、骨钙蛋白(osteocalcin,OC)及BMP-2表达;1、7、14、21 d行茜素红染色观察。取3月龄雄性SD大鼠24只,建立8 mm颅骨骨缺损模型,随机分为4组(n=6),分别植入对应的4组支架材料,术后8周取材行HE染色观察骨缺损修复情况,并检测新生骨体积百分比和新生血管密度。结果 B、C、D组抗弯强度、断裂功、弹性模量及硬度均显著高于A组,C、D组高于B组,D组高于C组,差异均有统计学意义(P<0.05)。CCK-8法检测示随培养时间增加,细胞活性逐渐增加,3、7、14 d时B、C、D组A值均显著高于A组,C、D组高于B组(P<0.05),C、D组间比较差异无统计学意义(P>0.05)。Live/Dead荧光染色示,7、14 d时B、C、D组活细胞比例均显著高于A组(P<0.05),7 d时C、D组显著高于B组(P<0.05);RT-PCR检测示,7、14 d时B、C、D组各基因相对表达量均显著高于A组,C、D组显著高于B组(P<0.05),C、D组间比较差异无统计学意义(P>0.05);茜素红染色示随培养时间延长,各组支架材料表面的红色钙盐沉积均逐渐加深变厚,14、21 d时B、C、D组A值均显著高于A组,C、D组高于B组(P<0.05),C、D组间差异无统计学意义(P>0.05)。动物Objective To investigate the mechanical properties of the novel compound calcium phosphate cement（CPC） biological material as well as the biological activity and osteogenesis effects of induced pluripotent stem cells（i PS） seeding on scaffold and compare their bone regeneration efficacy in cranial defects in rats. Methods According to the different scaffold materials, the experiment was divided into 4 groups： pure CPC scaffold group（group A）,CPC∶10%wt chitosan as 2∶1 ratio mixed scaffold group（group B）, CPC∶10%wt chitosan∶whisker as 2∶1∶1 ratio mixed scaffold group（group C）, and CPC∶10%wt chitosan∶whisker as 2∶1∶2 ratio mixed scaffold group（group D）.Mechanical properties（bending strength, work-of-fracture, hardness, and modulus of elasticity） of each scaffold were detected. The scaffolds were cultured with fifth generation i PS-mesenchymal stem cells（MSCs）, and the absorbance（A）values of each group were detected at 1, 3, 7, and 14 days by cell counting kit 8（CCK-8） method; the alkaline phosphatase（ALP） activity, Live/Dead fluorescence staining and quantitative detection, ALP, Runx2, collagen typeⅠ, osteocalcin（OC）, and bone morphogenetic protein 2（BMP-2） gene expressions by RT-PCR were detected at 1, 7, and14 days; and the alizarin red staining were detected at 1, 7, 14, and 21 days. Twenty-four 3-month-old male Sprague Dawley rats were used to establish the 8 mm-long skull bone defect model, and were randomly divided into 4 groups（n=6）; 4 kinds of scaffold materials were implanted respectively. After 8 weeks, HE staining was used to observe the repair of bone defects and to detect the percentage of new bone volume and the density of neovascularization. Results The bending strength, work-of-fracture, hardness, and modulus of elasticity in groups B, C, and D were significantly higher than those in group A, and in groups C, D than in group B, and in group D than in group C（P〈0.05）. CCK-8 assay showed that cell activity gradually i

关 键 词：磷酸钙骨水泥 壳聚糖 胡须 诱导多能干细胞 骨组织工程 复合生物材料 

分 类 号：R318.08[医药卫生—生物医学工程]