多孔CPC组织工程肋骨支架制备及BMSCs在其表面增殖黏附的实验研究  

PREPARATION OF CPC FOR TISSUE ENGINEERING ARTIFICIAL RIB AND A STUDY ON PROLIFERATION AND ADHESION OF BMSCs ON CPC

作　　者：唐华[1] 徐志飞[1] 刘昌胜[2] 秦雄[1] 吴彬[1] 乌立晖[1] 

机构地区：[1]第二军医大学附属长征医院胸心外科,上海200003 [2]华东理工大学医用生物材料研究中心

出　　处：《中国修复重建外科杂志》2008年第10期1238-1241,共4页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30571847)~~

摘　　要：目的通过形态学观察、细胞黏附和细胞增殖实验观察多孔CPC材料作为组织工程肋骨支架的可行性。方法取幼猪骨髓分离培养BMSCs并传代,另自制5mm×5mm×5mm大小的多孔CPC材料,micro-CT观察孔径、孔隙率及羟基磷灰石(hydroxyapatite,HA)含量,并与人正常松质骨作对比。取24孔培养板,于孔内置入多孔CPC材料1块,共15块,调整第1代BMSCs浓度为6×105个/mL,将细胞悬液150μL滴加到多孔CPC材料上,置于孵育箱中复合培养4、12、24h后,收集细胞并计数,计算细胞黏附率;行倒置相差显微镜观察、MTT法检测多孔CPC材料表面BMSCs的生长情况;扫描电镜观察接种BMSCs7d的多孔CPC材料,并与单纯多孔CPC材料和正常人松质骨对比。结果BMSCs与多孔CPC材料复合培养4、12、24h的细胞黏附率分别为28.00%±0.98%、46.70%±1.14%和48.50%±1.18%。MTT法检测细胞与材料复合培养1、2、3、4、5、6d的吸光度(A)值分别为1.103±0.214、1.557±0.322、1.920±0.178、2.564±0.226、2.951±0.415和3.831±0.328,呈明显上升趋势。倒置相差显微镜下可见细胞生长旺盛,贴壁良好,未出现毒性反应。micro-CT检查示多孔CPC材料的孔径均衡,且互相连通,HA含量为(1101.2228±0.6184)mg/ccm,孔隙率为70.26%±0.45%;人正常松质骨HA含量为(1072.5523±0.7442)mg/ccm,孔隙率为72.82%±0.51%;两组比较差异无统计学意义(P>0.05)。扫描电镜观察单纯多孔CPC材料孔径与正常人松质骨孔径大小、结构相似,且互相连通;BMSCs在多孔CPC材料表面排列有序,细胞呈片状,部分呈簇状,细胞形态呈多边形,细胞间可见较多细胞间质。结论多孔CPC材料结构和成分均接近正常人松质骨,BMSCs在其表面生长良好,适合作为组织工程肋骨支架材料,但其对细胞的黏附性有待进一步改善。Objective To access the possibility of CPC as a suitable scaffold for tissue morphologic observation, adhesion experiments and cellar proliferation experiments. Methods engineering artificial rib by The 5mm×5mm×5mm CPCs were prepared and the structure and components of CPC were compared with those of the normal human bone by micro-CT and scanning electron microscope. Bone marrow aspirates were harvested from the young pig and monuclear cells were separated. The first passage ceils were collected and re-suspended in the culture media at a density of 6 × 10^5 cells/mL. There was 150 μL suspension which was incoluated on the CPC, and then cells were recollected and counted 4, 12 and 24 hours after inoculation. MTT was used to examine the growth condition of BMSCs on the surface of CPC. The scanning electron microscope was used to observe the CPC scaffold 7 days after inoculation, and comparison was made with CPC and the normal human bone. Results The adhesion rate of CPC was 28.00% ± 0.98%, 46.70% ± 1.14% and 48.50% ± 1.18%, respectively 4, 12 and 24 hours after compound culture. The proliferation rate of CPC was 1.103 ± 0.214, 1.557 ± 0.322, 1.920 ± 0.178, 2.564 ± 0.226, 2.951 _± 0.415 and 3.831 ± 0.328, respectively 1, 2, 3, 4, 5 and 6 days after compound culture, with an obvious rising trend. The micro-CT demonstrated that the content of hydroxyapatite of porous phosphate calcium was （1 101.222 8 ± 0.618 4） mg/ccm while that of the normal human bone was （1 072.552 3 ± 0.744 2） mg/ccm, and the porosity of porous phosphate calcium was 70.26% ± 0.45% while that of the normal human bone was 72.82% ± 0.51%, and there was no significant difference （P 〉 0.05）. The experiment of cell proliferation showed that the cell which was cultivated with porous phosphate calcium proliferated rapidly. Through the inverted phase contrast microscope, it was found that the cells grew well and there was no dead cell, which indicated that the material had no toxicity. The rate of the cell adhesion to CPC wa

关 键 词：组织工程肋骨 多孔CPC材料 BMSCS 增殖黏附 

分 类 号：R318[医药卫生—生物医学工程] Q813[医药卫生—基础医学]