Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair
Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair
复制标题
通过多光子显微镜可视化体内转基因 3D-PLGA/nHAp 支架中的血管生成，用于修复颅骨关键骨缺损
DOI：
10.3791/55381
复制
发表时间：
2017-09-01
影响因子：
1.2
通讯作者：
Ren, Pei-Gen
中科院分区：
文献类型：
--
作者：
Li, Jian;Jahr, Holger;Ren, Pei-Gen
严重骨缺损的重建仍然是一个严重的临床问题，因为修复过程中组织工程支架内的血管生成不良，导致缺乏足够的血液供应并导致新组织坏死。快速血管化是新组织存活和与现有宿主组织整合的重要先决条件。支架中脉管系统的从头生成是使骨再生更有效、允许修复组织生长成支架的最重要步骤之一。为了解决这个问题，生物材料支架的基因修饰被用来加速血管生成和骨生成。然而，在三维（3D）支架或新骨组织中实时可视化和跟踪体内血管形成仍然是骨组织工程的一个障碍。多光子显微镜（MPM）是一种新型生物成像方式，可以以高分辨率和微创方式从生物结构中获取体积数据。本研究的目的是通过多光子显微镜观察体内转基因 3D-PLGA/nHAp 支架中血管生成的情况，以修复颅骨关键骨缺损。 PLGA/nHAp 支架被功能化，用于持续递送携带慢病毒载体 (LV-pdgfb) 的生长因子 pdgf-b 基因，以促进血管生成并增强骨再生。在植入支架的颅骨严重骨缺损小鼠模型中，PHp 支架中的血管面积 (BVA) 显着高于 PH 支架中的血管面积 (BVA)。此外，pdgf-b和血管生成相关基因vWF和VEGFR2的表达也相应增加。 MicroCT 分析表明，与其他组相比，PHp 组的新骨形成显着改善。据我们所知，这是首次将多光子显微镜用于骨组织工程，以在体内实时研究 3D 生物可降解支架中的血管生成。
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.