基于血管化机制构建萎缩型骨不连类器官芯片与初步实验研究  

Construction and experimental research of atrophic bone nonunion organoid on chip based on vascularization mechanism

作　　者：胡衍 张浩[2] 刘晗[2] 周晨阳 刘金龙[2] 李啸群 崔进 周启荣 王晓林[3] 陈晓[1,2] 王栋梁[1] 苏佳灿 Hu Yan;Zhang Hao;Liu Han;Zhou Chenyang;Liu Jinlong;Li Xiaoqun;Cui Jin;Zhou Qirong;Wang Xiaolin;Chen Xiao;Wang Dongliang;Su Jiacan(Department of Orthopaedic Surgery,Xinhua Hospital Affiliated to Shanghai Jiao Tong University Shcool of Medicine,Shanghai 200092,China;Institute of Translational Medicine,Shanghai University,Shanghai 200444,China;School of Electronic Information and Electrical Engineering,Shanghai Jiao Tong University,Shanghai 200240,China;Department of trauma orthopaedic surgery,Changhai Hospital,Shanghai 200233,China)

机构地区：[1]上海交通大学医学附属新华医院骨科,上海200092 [2]上海大学转化医学研究院,上海200444 [3]上海交通大学电子信息与电气工程学院,上海200240 [4]上海长海医院创伤骨科,上海200233

出　　处：《中华骨科杂志》2023年第24期1673-1680,共8页Chinese Journal of Orthopaedics

基　　金：国家自然科学基金项目(82302397);上海市科技创新行动计划实验动物研究领域项目(23141900600)。

摘　　要：目的构建基于血管化机制的骨不连类器官芯片,并探讨无菌性骨不连发生机制。方法设计半开放微流控芯片,实现人骨髓间充质干细胞(bone marrow mesenchymal stromal cells,BMSC)、人胚肺成纤维细胞(human fetal lung fibroblast 1,HFL1)与人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)共培养,建立三维器官芯片体系。设置HFL1与HUVEC不同比例与BMSC共培养,分为对照组(HFL1∶HUVEC=1∶1)、纤维化组(HFL1∶HUVEC=3∶1)与血管化组(HFL1∶HUVEC=1∶3)。通过碱性磷酸酶(alkaline phosphatase,ALP)、茜素红(alizarin red)染色观察BMSC成骨分化情况,qPCR分析成骨标志基因SP7、RUNX2、ALPL、BGLAP及血管化相关基因KDR、VWF转录情况,Western blot检测成骨标志蛋白RUNX2和ALP表达水平。结果BMSC、HFL1和HUVEC共培养体系中BMSC正常生长且出现明显生物矿化行为,血管内皮细胞能够形成有序血管结构,证实体系构建成功。相较对照组,纤维化组BMSC成骨分化下降趋势不明显,矿化标志基因ALPL和BGLAP相对表达量为0.55±0.19、0.42±0.27,差异均有统计学意义(P<0.05);血管化组基因KDR和VWF下调,相对表达量为0.49±0.17、0.49±0.21,差异均有统计学意义(P<0.05)。相较对照组,血管化组BMSC成骨分化基因SP7、RUNX2、ALPL和BGLAP上调,相对表达量分别为2.91±0.52、3.83±1.87、3.22±1.29和5.21±1.46,差异均有统计学意义(P<0.05);血管化基因KDR、VWF上调,相对表达为8.24±2.84、5.32±1.67,差异均有统计学意义(P<0.05)。Western blot结果表明RUNX2、ALP在血管化组中表达增加,纤维化组中表达减少。结论骨不连类器官芯片能够部分模拟骨不连局部微环境,纤维化可能导致骨形成能力和血管化水平显著下降,是无菌性骨不连发生的潜在重要原因。Objective To design and construct a bone nonunion organoid on chip and explore the mechanism of aseptic bone nonunion.Methods First a semi-open microfluidic chip was designed,on which human bone marrow mesenchymal stromal cells(BMSC),human fetal lung fibroblast 1,(HFL1)and human umbilical vein endothelial cells(HUVEC)were co-cultured,and a three-dimensional organ on chip system was established.Different proportions of HFL1 and HUVEC were co-cultured with BMSC,which were divided into the control group(HFL1∶HUVEC=1∶1),the fibrosis group(HFL1∶HUVEC=3∶1)and the vascularization group(HFL1∶HUVEC=1∶3).The osteogenic differentiation of BMSC was observed by alkaline phosphatase(ALP)and Alizarin red staining.The transcription level of osteogenic marker genes SP7,RUNX2,ALPL,and BGLAP,and vascularization related genes KDR and VWF were analyzed by qPCR.The expression levels of RUNX2 and ALP were determined by Western Blot.Results In the co-culture system of BMSCs,HFL1,and HUVECs,BMSCs exhibited normal growth and apparent biomineralization behavior.Endothelial cells were capable of forming structured vascular networks,confirming the successful establishment of the system.Compared to the baseline group,the fibrotic group showed no significant decrease in BMSC osteogenic differentiation.The relative expression levels of the mineralization marker genes ALPL and BGLAP were 0.55±0.19(P<0.001)and 0.42±0.27(P<0.001),respectively.Vascularization genes KDR and VWF were downregulated,with relative expression levels of 0.49±0.17(P<0.001)and 0.49±0.21(P<0.001).In contrast,in the vascularized group,BMSC osteogenic differentiation genes SP7,RUNX2,ALPL,and BGLAP were upregulated,with relative expression levels of 2.91±0.52(P<0.001),3.83±1.87(P<0.001),3.22±1.29(P<0.001),and 5.21±1.46(P<0.001),respectively.Vascularization genes KDR and VWF were also upregulated,with relative expressions of 8.24±2.84(P<0.001)and 5.32±1.67(P<0.001).Western blot results indicated increased expression of RUNX2 and ALP in the vascularized grou

关 键 词：骨折 不愈合 类器官 新生血管化 病理性 器官芯片 

分 类 号：R68[医药卫生—骨科学]