成骨分化后的去分化BMSCs移植对前十字韧带重建腱骨界面骨形成的作用及机制  

The promotion of bone formation at the tendon-bone interface after ACL reconstruction with De-BMSCs transplantation and its mechanism

作　　者：铁楷[1] 蔡竞航 秦俊[1] 肖浩[1] 上官杨帆 陈廖斌[1] Tie Kai;Cai Jinghang;Qin Jun;Xiao Hao;Shangguan Yangfan;Chen Liaobin(Department of Orthopedic Surgery,Zhongnan Hospital of Wuhan University,Wuhan 430071,China)

机构地区：[1]武汉大学中南医院骨科,武汉430071

出　　处：《中华骨科杂志》2022年第8期519-529,共11页Chinese Journal of Orthopaedics

基　　金：国家自然科学基金青年项目(81702159)。

摘　　要：目的观察成骨分化后的去分化骨髓间质干细胞(differentiation osteogenic bone marrow mesenchymal stem cells,De-BMSCs)移植对前十字韧带(anterior cruciate ligament,ACL)重建术后腱骨界面骨形成的促进作用,并探索De-BMSCs成骨分化效应增强的分子机制。方法从家兔胫骨和股骨中提取BMSCs,经成骨诱导分化后用普通培养基进行去分化培养制备De-BMSCs,鉴定De-BMSCs的干细胞特性。建立家兔ACL重建模型,分为3组:腱骨界面注射海藻酸钠凝胶(对照组);腱骨界面注射复合BMSCs海藻酸钠凝胶(BMSCs组);腱骨界面注射复合De-BMSCs海藻酸钠凝胶(De-BMSCs组)。术后4周和12周取膝关节标本,进行组织学、影像学、生物力学检测,评估腱骨界面骨形成情况。De-BMSCs体外成骨诱导分化,给予活化T细胞核转录因子1(nuclear factor of activated T cells 1,NFATc1)RNA干扰处理,检测成骨分化标志基因及Nanog/NFATc1/Osterix信号通路的表达,明确De-BMSCs成骨分化效应增强的分子机制。结果De-BMSCs可向成骨、成脂、成软骨分化,具有干细胞特性(均P<0.05)。术后4周De-BMSCs组BV/TV值为0.36±0.01,较对照组0.24±0.03和BMSCs组0.30±0.02明显增加(P<0.05),最大负荷(40.34±1.19)N和刚度(20.67±2.14)N/mm较对照组[(14.88±2.74)N,(8.67±2.19)N/mm]和BMSCs组[(26.31±1.76)N,(13.81±2.14)N/mm]明显升高(均P<0.05);术后12周De-BMSCs组BV/TV值0.47±0.02较对照组0.30±0.02和BMSCs组0.35±0.03明显增加(均P<0.05),最大负荷(64.46±6.69)N和刚度(25.18±3.11)N/mm较对照组[(41.01±6.12)N,(11.59±2.54)N/mm]和BMSCs组[(48.21±4.12)N,(15.89±2.94)N/mm]明显升高(均P<0.05);De-BMSCs成骨分化过程中,Nanog的表达增加(P<0.05),NFATc1的表达上升且其与Osterix的相互作用增强(P<0.05),成骨分化标志基因COL1A、OCN、OPN的表达增加(均P<0.05)。结论De-BMSCs移植可促进ACL重建后的腱骨界面骨形成,其作用机制可能是Nanog/NFATc1/Osterix信号通路介导了De-BMSCs成骨分化效应增强。Objective This study aimed to investigate the effect of differentiation osteogenic bone marrow mesenchymal stem cells(De-BMSCs)transplantation on the promotion of bone formation at the tendon-bone interface after anterior cruciate ligament reconstruction(ACLR),and further explored the molecular mechanism of the enhanced osteogenic effect of De-BMSCs.Methods BMSCs from femur and tibia of New Zealand White rabbit were subjected to osteogenic induction and then cultured in no osteogenic factor medium;the obtained cell population was termed De-BMSCs.De-BMSCs were induced into osteo-,chondro-and adipo-differentiation in vitro to examine the characteristics of primitive stem cells.ACLR model with a semitendinosus tendon were performed in 48 adult rabbits,three groups were established:control group with alginate gel injectionat the tendon-bone interface,BMSCs group with the injection of alginate gel containing BMSCs,De-BMSCs group with the injection of alginate gel containing De-BMSCs.At 4 and 12 weeks after surgery,rabbits in each group were sacrificed to evaluate tendon-bone healing by histologic staining,micro-CT examination,and biomechanical test.During osteogenic differentiation of De-BMSCs,si-RNA of nuclear factor of activated T cells 2(NFATc2)si-RNA of nuclear factor of activated T cells 1(NFATc1)were used to verify the molecular mechanism of enhanced osteogenic effect of De-BMSCs.Results De-BMSCs exhibited some properties similar to BMSCs including multiple differentiation potential and cell surface marker.At 4 weeks after surgery,the BV/TV value of the De-BMSCs group 0.36±0.01 was significantly higher than that of the control group 0.24±0.03 and BMSCs group 0.30±0.02(all P<0.05),and the maximum load 40.34±1.19 N and stiffness 20.67±2.14 N/mm were significantly higher than those in the control group 14.88±2.74N,8.67±2.19 N/mm and the BMSCs group 26.31±1.76 N,13.81±2.14 N/mm(all P<0.05).At 12 weeks after surgery,the BV/TV value of the De-BMSCs transplantation group 0.47±0.02 was significantly higher tha

关 键 词：细胞分化 细胞去分化 间质干细胞 前交叉韧带 

分 类 号：R687.4[医药卫生—骨科学]