Vascularization of critical-sized craniomaxillofacial defects

项目摘要 缺乏适当的血管化导致治疗临界尺寸颅颌面畸形的最终失败 缺陷缺损的大尺寸阻碍了血液成分从周围渗透 环境进入缺损的内部，从而阻碍血管形成。在这种情况下，血管 内皮生长因子（VEGF）是能够重建组织氧供应的最有效的因子。 虽然应用外部VEGF是在临界尺寸缺损中形成血管的关键手段，但其轻微的 不受控制的给药是有风险的并且可能是致瘤的。因此，常规方法无法用于 封装和递送VEGF。在这项提案中，我们将开发一种新的芯片上方法来递送VEGF 使用微流体平台具有精确和持续的释放能力。我们新颖的设计使得 以高度可控和可重复的方式生产单分散颗粒，为我们提供精细的 调整颗粒的大小、微观结构、负载能力和释放速率，以及平衡pH值和 维持VEGF的生物活性。VEGF的释放不仅必须受到控制和持续，而且还必须高度依赖于血管内皮细胞。 当将加载VEGF的颗粒移动到不需要的区域中时， 而且可能有风险因此，在另一种策略中，负载VEGF的颗粒将被固定在新的3D打印的载体上。 专门为临界尺寸缺陷设计的支架。这种新型支架的设计（已申请专利） 受钢筋混凝土的启发，其中钢筋被嵌入到主体材料中，以增强 支架的机械性能（提高100-375倍）。换句话说，它是一种混合支架， 由两部分组成：1）骨架钢筋：无孔和缓慢生物降解的成分承担 支架的机械必要性，和2）主体成分：多孔和快速生物降解的成分 承担支架的生物必需品。虽然钢筋的机械强度是 这使得支架适合于临界尺寸的缺陷，这是Rebar的另一个功能， 可降解性（6个月），使得该设计成为VEGF递送目的的完美选择。钢筋将提供 让我们有机会将载有VEGF的颗粒固定在固体表面上， 其他地方固定过程本身是我们实验室开发的一种新方法，可以牢固地将这些 颗粒附着在支架的钢筋上。加载VEGF的支架将进行详细的体外分析， 在生物反应器内释放调节，这可以模拟身体状况。VEGF释放曲线将是 调整以达到目标值（1.2 ng/ml/天/cm 3支架），并进行全面的体外分析 将评估构建体的成骨和血管生成特征。优化的VEGF负载支架 将使用新西兰白色兔的临界尺寸牙槽骨缺损进行详细的体内研究。的 新骨形成和血管生成将被充分研究，以评估VEGF负载支架的功能 与不含VEGF的支架相比，以及用目前的治疗方式治疗的缺陷。

Project Summary Lack of proper vascularization leads to the ultimate failure in treatment of critical-sized craniomaxillofacial defects. The large size of the defect obstructs penetration of blood components from the surrounding environment into the inner parts of the defect, and thus hinders vascularity. In such situations, vascular endothelial growth factor (VEGF) is the most effective factor that can reestablish the oxygen supply to tissues. While applying external VEGF is a key means for blood vessel formation in critical-sized defects, its slight uncontrolled administration is risky and can be tumorigenic. Thus, conventional methods cannot be used for encapsulation and delivery of VEGF. In this proposal, we will develop a new on-chip method for delivery of VEGF with precise and sustained release capabilities using a microfluidic platform. Our novel design allows making monodispersed particles in a highly controllable and reproducible manner, providing us with the ability to fine- tune the size, microstructure, loading capacity and release rate of particles, in addition to balancing the pH and maintaining the VEGF bioactivity. Release of VEGF must not be only controlled and sustained, but also highly localized in the region of the defect as moving the VEGF-loaded particles into unwanted areas is not favorable and can be risky. Thus, in another strategy, the VEGF-loaded particles will be immobilized onto a new 3D-printed scaffold specifically designed for critical-sized defects. The design of this novel scaffold (filed for patent) is inspired by reinforced concrete, in which reinforcing Rebars are embedded in the host material to enhance the mechanical properties of the scaffold (100-375 times improvement). In other words, it is a hybrid scaffold, made of two components: 1) Skeleton Rebars: non-porous and slowly-biodegradable constituent undertaking mechanical necessities of the scaffold, and 2) Host Component: porous and rapidly-biodegradable constituent undertaking biological necessities of the scaffold. Although the mechanical strength of Rebars is the property that makes the scaffold appropriate for critical-sized defects, another functionality of the Rebar, which is its slow degradability (6 months), makes the design a perfect choice for the VEGF delivery purpose. Rebars will provide us with the opportunity to immobilize VEGF-loaded particles on a solid surface and not let the particles move elsewhere. The immobilization process itself is a new method developed in our lab that can firmly attach these particles to the rebars of the scaffolds. The VEGF-loaded scaffold will undergo a detailed in vitro analysis and release adjustment inside a bioreactor, which can mimic the body condition. The VEGF release profiles will be adjusted to reach the target value (1.2 ng/ml per day per cm3 of scaffold), and the comprehensive in vitro analyses will evaluate the osteogenesis and angiogenesis characters of the construct. The optimized VEGF-loaded scaffold will undergo a detailed in vivo study using critical-sized alveolar bone defects in New Zealand white rabbits. The new bone formation and angiogenesis will be fully studied to assess the functionality of the VEGF-loaded scaffold in comparison with a VEGF-free scaffold, as well as defects treated with a current therapeutic modality.