Endothelial cell specification at the osteogenic and angiogenic interface in cranial bone tissue engineering

摘要 修复和重建因肿瘤切除、创伤和感染而造成的骨丢失仍然是一个重要的 临床挑战。全世界大约有300万人使用自体或同种异体骨移植 每年进行手术，其中6%是颅颌面性质的。骨组织工程学受到欢迎 作为替代自体骨移植修复骨缺损的最终解决方案。然而，长期的成功 骨组织工程的发展因工程化结构的血管化不足而受阻。海流 组织工程支架的血管化缺乏进展归因于我们对其认识的不完全 血管生成和血管床在骨修复和再生中的作用。功能良好的血管网络由 动脉、静脉和连接动脉和静脉的微血管的毛细血管接口 灌流。而动脉和静脉内皮细胞的特性在早期已经得到了很好的研究 胚胎发育、动静脉扩张的后天调节及毛细血管的规范 修复和再生过程中的水平还知之甚少。最近的一系列研究表明， 低氧影响兔成骨和血管生成界面的内皮细胞(EC)规格 发育和衰老。低氧诱导因子-1(HIF-1)途径的显著遗传操纵 影响特定的毛细血管亚群的形成，称为H型(CD31HighEmcnHigh)血管 在长骨干骺端与OSX+成骨细胞偶联。为了更好地理解 低氧在成骨和血管生成界面的修复和再生中，我们建立了一系列新的 能够对毛细血管进行高分辨率、定量和功能分析的成像方法 在颅骨缺损处与Col(I)2.3 GFP+成骨细胞偶联。利用这些新的成像方法 一个逐层启用的纳米纤维介导的颅骨缺损修复模型，我们证明了成骨- 依赖性血管生成由形态和功能不同的CD31+Emcn+和CD31+Emcn-组成 船只。血管类型分布和骨再生检查显示不同 CD31+Emcn+和CD31+Emcn-血管的血管生成反应及分布对比 Col I(2.3)GFP+成骨细胞、新骨和非骨形成组织，提示EC在 在骨修复和再生过程中，毛细血管水平是成骨依赖的血管生成的关键组成部分。 基于这些发现，我们建议研究低氧对EC规范的影响以及 EC规范在颅骨缺损修复和再生过程中骨形成的失调。三 互补的AIMS将结合成像、遗传和工程方法来定义成骨- 依赖EC规范和低氧在修复和再生中的作用。我们的研究的成功将会 为修复中的成骨和血管生成机制提供了新的见解，潜在地提供了新的 骨再生的翻译靶点。

Abstract Repair and reconstruction of bone loss due to tumor resection, trauma and infection remains a significant clinical challenge. Worldwide, autografts or allografts are used in approximately 3 million orthopaedic procedures annually, of which 6% are craniomaxillofacial in nature. Bone tissue engineering has been hailed as the ultimate solution for replacing bone autograft in repair of bone defects. However, the long-term success of bone tissue engineering is impeded by inadequate vascularization of the engineered construct. The current lack of progress in vascularization of tissue engineered scaffold is attributed to our incomplete understanding of angiogenesis and vascular beds in bone repair and regeneration. A functional blood vessel network consists of arteries, veins and a capillary interface that connects arterial and venous microvessels for proper vascular perfusion. While the specification of arterial and venous endothelium has been well studied during early embryonic development, the postnatal regulation of arterial and venous expansion and specification at capillary level during repair and regeneration is poorly understood. A series of recent studies have suggested that hypoxia affects the endothelial cell (EC) specification at the osteogenic and angiogenic interface in development and aging. Genetic manipulation of the hypoxia inducible factor 1 (HIF-1) pathway markedly affects the formation of specific subsets of capillary vessels, termed Type H (CD31highEmcnhigh) vessels that couple to OSX+ osteoblasts at the long bone metaphysis. To gain a better understanding of the critical role of hypoxia at the osteogenic and angiogenic interface in repair and regeneration, we established a series of novel imaging approaches that permit high resolution, quantitative, and functional analyses of capillary vessels that couple to Col (I) 2.3 GFP+ osteoblasts at a cranial bone defect site. Utilizing these novel imaging approaches in a layer-by-layer enabled, nanofiber-mediated cranial defect repair model, we demonstrate that osteogenesis- dependent angiogenesis consists of morphologically and functionally distinct CD31+Emcn+ and CD31+Emcn- vessels. Examination of blood vessel type distribution and bone regeneration demonstrates differential angiogenic responses and contrasting distributions of CD31+Emcn+ and CD31+Emcn- vessels associated with Col I (2.3) GFP+ osteoblasts, new bone and non-bone forming tissue, suggesting that EC specification at the capillary level is a key component of osteogenesis-dependent angiogenesis in bone repair and regeneration. Based on these findings, we propose to examine the effects of hypoxia on EC specification and the impact of dysregulation of EC specification on bone formation during cranial defect repair and regeneration. Three complementary Aims will combine imaging, genetic and engineering approaches to defining the osteogenesis- dependent EC specification and the role of hypoxia in repair and regeneration. The success of our study will provide novel insights into mechanisms of osteogenesis and angiogenesis in repair, potentially offering novel translational targets for bone regeneration.