Li Jian, Jahr Holger, Zheng Wei, Ren Pei-Gen
Center for Translational Medicine Research and Development, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences.
Department of Orthopedic Surgery, Maastricht UMC+; Department of Orthopaedic Surgery, University Hospital RWTH.
J Vis Exp. 2017 Sep 7(127):55381. doi: 10.3791/55381.
The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.
由于在修复过程中组织工程支架内血管生成不良，导致血液供应不足并引起新组织坏死，因此临界尺寸骨缺损的修复仍然是一个严重的临床问题。快速血管化是新组织存活并与现有宿主组织整合的重要前提。支架中血管的从头生成是提高骨再生效率的最重要步骤之一，它能使修复组织长入支架。为了解决这个问题，人们利用生物材料支架的基因改造来加速血管生成和成骨。然而，实时三维（3D）可视化和追踪生物材料支架或新骨组织中的体内血管形成仍然是骨组织工程面临的一个障碍。多光子显微镜（MPM）是一种新型生物成像技术，能够以高分辨率和微创方式从生物结构中获取体积数据。本研究的目的是利用多光子显微镜在体内可视化经基因改造的3D-PLGA/nHAp支架修复颅骨临界骨缺损时的血管生成情况。PLGA/nHAp支架经过功能化处理，用于持续递送携带血小板衍生生长因子B基因（pdgf-b）的慢病毒载体（LV-pdgfb），以促进血管生成并增强骨再生。在支架植入的颅骨临界骨缺损小鼠模型中，PHp支架中的血管面积（BVA）显著高于PH支架。此外，pdgf-b以及血管生成相关基因vWF和VEGFR2的表达相应增加。显微CT分析表明，与其他组相比，PHp组的新骨形成有显著改善。据我们所知，这是首次将多光子显微镜用于骨组织工程，以实时体内研究3D生物可降解支架中的血管生成情况。
