联合培养血管内皮细胞与脂肪干细胞构建组织工程骨异位成骨

Heterotopic osteogenesis study of tissue engineered bone by co-culture of vascular endothelial cells and adipose-derived stem cells

目的探讨将联合培养血管内皮细胞(vascular endothelial cells,VECs)和脂肪干细胞(adiposederived stem cells,ADSCs)的培养体系作为种子细胞的组织工程骨异位成骨能力。方法采用纤维粘连蛋白复合部分脱蛋白骨(partially deproteinised bone,PDPB)制备部分脱蛋白生物骨(partially deproteinised biologic bone,PDPBB);分离培养18周龄SD大鼠ADSCs及足月妊娠SD大鼠脐血VECs。体外构建3种组织工程骨:PDPBB+VECs(A组)、PDPBB+ADSCs(B组)、PDPBB+联合培养细胞(VECs∶ADSCs为1∶1,C组),以单纯PDPBB为对照组(D组)。细胞移植后10 d行扫描电镜观察细胞在各组支架上的黏附情况。取18周龄SD大鼠48只,随机分为A、B、C、D 4组,每组12只。分别将A、B、C、D 4种支架材料植入相应组别的大鼠股部肌袋,术后2、4、8、12周处死动物行大体观察及HE染色和Masson染色组织学观察,并取Masson染色切片行骨胶原量定量检测。结果扫描电镜观察示,PDPB材料孔隙内部相互连通;纤维粘连蛋白修饰的PDPBB表面大量片状蛋白结晶松散附着于支架表面;复合细胞联合培养10 d后,C组大量细胞与PDPBB附着,细胞相互堆积形成细胞团,多角形细胞和梭形细胞混合分布,细胞沿骨小梁生长,形成细胞层。大体观察示术后2周各组肉芽组织开始长入材料孔隙。C组自4周后材料表面逐渐产生大量白色软骨样物质,表面高低不平。至12周时,A组材料表面血管量增多,材料呈实变;B组材料表面出现少许白色软骨样物质,但材料孔隙未见明显减小;D组材料孔隙大小未见明显减小。组织学观察可见术后2周,各组间骨胶原量比较差异无统计学意义(F=2.551,P=0.088);术后4、8、12周,C组骨胶原量显著高于其余3组,B组高于D组,差异均有统计学意义(P<0.05);A组与B、D组比较差异均无统计学意义(P>0.05)。结论联合培养VECs与ADSCs作为种子细胞构建组织工程骨的异位成骨能力最强。

Objective To investigate the heterotopic osteogenesis of tissue engineered bone using the co-culture system of vascular endothelial cells(VECs) and adipose-derived stem cells(ADSCs) as seed cells. Methods The partially deproteinized biological bone(PDPBB) was prepared by fibronectin combined with partially deproteinized bone(PDPB).The ADSCs of 18-week-old Sprague Dawley(SD) rats and VECs of cord blood of full-term pregnant SD rats were isolated and cultured. Three kinds of tissue engineered bone were constructed in vitro: PDPBB+VECs(group A), PDPBB+ADSCs(group B), PDPBB+co-cultured cells(VECs∶ADSCs was 1∶1, group C), and PDPBB was used as control group(group D). Scanning electron microscopy was performed at 10 days after cell transplantation to observe cell adhesion on scaffolds.Forty-eight 18-week-old SD rats were randomly divided into groups A, B, C, and D, with 12 rats in each group. Four kinds of scaffolds, A, B, C, and D, were implanted into the femoral muscle bags of rats in corresponding groups. The animals were killed at 2, 4, 8, and 12 weeks after operation for gross observation, HE staining and Masson staining histological observation, and the amount of bone collagen was measured quantitatively by Masson staining section. Results Scanning electron microscopy showed that the pores were interconnected in PDPB materials, and a large number of lamellar protein crystals on the surface of PDPBB modified by fibronection were loosely attached to the surface of the scaffold. After 10 days of co-culture PDPBB and cells, a large number of cells attached to PDPBB and piled up with each other to form cell clusters in group C. Polygonal cells and spindle cells were mixed and distributed, and some cells grew along bone trabeculae to form cell layers. Gross observation showed that the granulation tissue began to grow into the material pore at2 weeks after operation. In group C, a large number of white cartilage-like substances were gradually produced on the surface of the material after 4 weeks, and the surface of the material was uneven. At 12 weeks, the amount of blood vessels on the surface of group A increased, and the material showed consolidation;there was a little white cartilage-like material on the surface of group B, but the pore size of the material did not decrease significantly;in group D, the pore size of the material did not decrease significantly. Histological observation showed that there was no significant difference in the amount of bone collagen between groups at 2 weeks after operation(F=2.551, P=0.088);at 4, 8, and 12 weeks after operation, the amount of bone collagen in group C was significantly higher than that in other 3 groups, and that in group B was higher than that in group D(P<0.05);there was no significant difference between group A and groups B, D(P>0.05). Conclusion The ability of heterotopic osteogenesis of tissue engineered bone constructed by co-culture VECs and ADSCs was the strongest.