三维打印MBG支架负载iPS-MCSs来源外泌体激活AMPK-ERK1/2信号通路治疗大段骨缺损

The reconstruction of large segmental bone defects is a major treatment challenge in orthopedic surgery, while recent studies have indicated that exosomes derived from stem cells provide a potent therapeutic option for injured tissue repair similar to stem cell transplantation. Importantly, direct exosomes therapy may overcome the limitations and risks associated with stem cell transplantation. 3D printing of MBG/PHBHHx(MPH) scaffold mimicking segmental long bone diaphysis structure also offers a perfect material to fill in the bone defect area. Based on previous studies, we found that exosomes derived from iPS-MSCs activated the AMPK-ERK1/2 signal pathway in BMSCs and effectively promoted bone defects repair in vivo. Furthermore, our microRNA array study proved that exosomes derived from iPS-MSCs contain some functional miRNAs targeting AMPK-ERK1/2 signal pathway. Hence, we plan to utilize a series of experiments to clarify the molecular mechanisms that how MPH scaffold combined with iPS-MSCs derived exosomes promote bone regeneration and how these functional microRNAs in exosomes activate AMPK-ERK1/2 signal pathway in BMSCs. This proposed study taking advantage of stem cell and 3D printing technique will provide new ideas and scientific basis for the treatment of segmental bone defects in future.

大段骨缺损的治疗一直是骨科医生面临的棘手问题，最新研究显示干细胞分泌的外泌体可发挥类似干细胞样的促组织修复作用，可避免干细胞移植带来的潜在风险，显示出广阔的应用前景。此外，利用三维打印技术制备长管状结构的MBG/PHBHHx（MPH）支架为大段骨缺损提供完美填充。我们在前期研究发现iPS-MSCs来源外泌体可有效修复骨缺损，基因芯片分析提示其可激活BMSCs的AMPK-ERK1/2信号通路，miRNA array分析提示其含有靶向该通路的关键功能性miRNAs。据此，本课题拟通过系列实验，进一步验证MPH 支架负载iPS-MSCs来源外泌体促进骨再生的生物学功能，筛选并明确MPH负载iPS-MSCs来源外泌体中促进骨再生的关键功能性miRNAs及其作用途径，阐明其通过激活AMPK-ERK1/2信号通路促进骨再生的分子机制。该课题的顺利实施，将为临床大段骨缺损的治疗提供新的思路和科学依据。

大段骨缺损的治疗一直是骨科医生面临的棘手问题，最新研究显示干细胞分泌的外泌体可发挥类似干细胞样的促组织修复作用，可避免干细胞移植带来的潜在风险，显示出广阔的应用前景。在本课题中我们利用三维打印技术制备长管状结构的MBG/PHBHHx（MPH）支架为大段骨缺损提供完美填充。将人诱导多能干细胞（human induced pluripotent stem cells，hiPSCs）定向诱导分化为间充质干细胞（mesenchymal stem cells，MSCs），分离提取其分泌的Exos（hiPS-MSC-Exos），体外实验分析其对骨再生重要前体细胞BMSCs的增殖、迁移、成骨以及成骨重要信号通路的影响。体内实验观察MPH负载iPS-MSCs来源外泌体（MPH-hiPS-MSC-Exos）促进骨再生修复的作用。实验结果显示超滤结合超离方法可成功提取Exos，电镜、粒径及标志物检测结果均显示hiPS-MSC-Exos符合Exos的特征；hiPS-MSC-Exos具有显著修复骨缺损的作用：组织学染色及免疫组化结果显示缺损处有大量新生组织长入并且血管化明显，micro-CT结果显示骨缺损处有明显新生骨长入，荧光标记结果显示hiPS-MSC-Exos加速骨盐沉积矿化；免疫组化结果显示hiPS-MSC-Exos促进成骨标志物的表达； hiPS-MSC-Exos可激活BMSCs的AMPK-ERK1/2信号通路促进其成骨功能：CCK8及划痕实验结果显示hiPS-MSC-Exos可显著促进BMSCs增殖与迁移，碱性磷酸酶活性检测结果显示hiPS-MSC-Exos可显著促进BMSCs成骨分化，基因表达谱芯片技术和生物信息学分析显示hiPS-MSC-Exos刺激BMSCs后AMPK-ERK1/2信号通路被显著激活，进一步特异通路阻断实验结果显示阻断AMPK-ERK1/2通路后hiPS-MSC-Exos促成骨发生效应被显著抑制。本研究该课题的顺利实施，将为临床大段骨缺损的治疗提供新的思路和科学依据。