miR⁃34a骨粉复合胶原基水凝胶促进辐照区骨缺损修复  

Combinational use of miR-34a functionalized bone powder with Col-Tgel enhances bone regeneration in irradiated bone defects

作　　者：刘欢 武曦 LIU Huan;WU Xi(Department of Stomatology,the Second Affiliated Hospital,Army Medical University,Chongqing 400037,China)

机构地区：[1]陆军军医大学第二附属医院口腔科,重庆400037

出　　处：《口腔疾病防治》2024年第9期674-683,共10页Journal of Prevention and Treatment for Stomatological Diseases

基　　金：国家自然科学基金青年科学基金项目(82001010);重庆市自然科学基金面上项目(stc2020jcyj-msxmX0638);陆军军医大学第二附属医院青年博士人才孵化计划(2023YQB042)。

摘　　要：目的探讨负载miR⁃34a的Bio⁃Oss■骨粉与转谷氨酰胺酶交联明胶(transglutaminase crosslinked gela⁃tin,Col⁃Tgel)的联合使用对辐照损伤大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)的成骨分化作用及对辐照区骨缺损修复的作用。方法本实验已获得单位实验动物伦理委员会批准。取2周龄SD大鼠长骨骨髓,培养BMSCs并进行鉴定。当BMSCs生长至贴满瓶底80%时,进行2 Gy剂量X线照射,制备BMSCs辐照损伤模型备用。将2.5、5μL Col⁃Tgel分别加入10 mg Bio⁃Oss■骨粉(P)中,制备复合骨替代材料PG⁃2.5和PG⁃5,通过体外和体内实验筛选骨粉与水凝胶合适比例。将lipofectamine 2000分别与Cy3⁃agomiR⁃34a、agomiR⁃34a或agomiR NC混合,然后将各组混合液分别加入10 mg Bio⁃Oss■骨粉(P)并进行冷冻干燥。将上述负载各组miR的10 mg Bio⁃Oss■骨粉和未负载miR的Bio⁃Oss■骨粉分别与2.5μL Col⁃Tgel混合,制备PG⁃Cy3⁃miR⁃34a、PG⁃miR⁃34a、PG⁃miR NC、PG组复合骨替代材料。将辐照后的BMSCs与PG⁃Cy3⁃miR⁃34a组复合骨替代材料共培养,使用共聚焦显微镜观察转染效果。将辐照后的BMSCs与PG⁃miR⁃34a组、PG⁃miR NC组、PG组复合骨替代材料共培养,使用RT⁃qPCR检测miR⁃34a表达、CCK⁃8检测细胞增殖,并在成骨诱导14 d后利用RT⁃qPCR检测成骨相关基因Runt相关转录因子2(Runt related transcription factor 2,Runx2)、碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin,OCN)的表达。取8周龄SD大鼠进行双侧胫骨15 Gy剂量X线照射,3周后在胫骨干骺端骨骺线下方2~3 mm处制备直径3 mm、深度2 mm的胫骨缺损,缺损区分别置入PG⁃miR⁃34a组、PG⁃miR NC组、PG组复合骨替代材料,植入8周后取材行micro⁃CT和HE切片观察体内骨缺损修复效果。结果2 Gy辐照影响BMSCs成骨分化能力,辐照组ALP染色浅于非辐照组,辐照组茜素红染色矿化结节少于非辐照组。10 mg Bio⁃OObjective To study the effect of the combinational use of miR-34a-functionalized Bio-Oss■bone powder with transglutaminase crosslinked gelatin(Col-Tgel)on the osteoblastic differentiation of bone marrow mesenchymal stem cells(BMSCs)and bone defect healing after irradiation.Methods The experiment was approved by the Animal Ethics Committee.BMSCs were isolated from the bone marrow of 2-week-old Sprague-Dawley(SD)rats and identified.After reaching 80%confluence,BMSCs were irradiated with 2 Gy of X-ray radiation to establish a radiation-damaged BMSC model for further experimentation.2.5μL or 5μL of Col-Tgel was mixed with 10 mg of Bio-Oss■(P)to prepare PG-2.5 and PG-5.The optimal proportion of Bio-Oss■(P)and Col-Tgel was determined through in vitro and in vivo experiments.Cy3-labeled agomiR-34a,agomiR-34a,or agomiR NC was mixed with lipofectamine 2000 and added to 10 mg of Bio-Oss■(P).The mixtures were lyophilized,and 2.5μL Col-Tgel was added to each group of lyophilized BioOss■/lipofectamine/miRNA complexes or to 10 mg of Bio-Oss■to obtain PG-Cy3-miR-34a,PG-miR-34a,PG-miR NC,and PG.Irradiated BMSCs were cocultured with PG-Cy3-miR-34a to evaluate cellular uptake of Cy3-agomiR-34a using confocal microscopy.Then,irradiated BMSCs were cocultured with PG-miR-34a,PG-miR NC,and PG.The expression of miR-34a was tested by RT-qPCR and cell proliferation was tested by CCK-8 assay.After 14 days of osteogenic induction,the mRNA expression of Runt-related transcription factor 2(Runx2),alkaline phosphatase(ALP),and osteocalcin(OCN)was tested by RT-qPCR.The bilateral tibias of 8-week-old SD rats were irradiated with a single dose of 15 Gy of X-ray radiation.Three weeks later,tibial defects with a diameter of 3 mm and a depth of 2 mm were created 2-3 mm below the epiphyseal line in the tibial metaphysis.The composite bone substitute materials of PG-miR-34a,PG-miR NC,and PG were implanted into the defect area.Eight weeks after implantation,the tibias were harvested and evaluated for bone regeneration using micro-CT ana

关 键 词：骨粉 骨髓间充质干细胞 成骨分化 骨修复 miR⁃34a 转谷氨酰胺酶交联明胶 辐照损伤 放疗 

分 类 号：R78[医药卫生—口腔医学]