Angiogenesis and Long-term Bone and Joint Allotransplant Survival

描述（由申请人提供）：由于原发和转移性肿瘤切除、先天性缺陷、创伤性丢失、感染或假体植入失败而引起的大量骨和关节缺损。现有的重建方法往往失败率很高。它们包括结构性同种异体移植物（感染、骨不连和应力性骨折）、带血管的自体移植物（可用性有限、大小/形状不匹配、发病率高）或假体置换（感染、假体周围骨折和失败）。这个难题需要一个更好的解决方案。活体同种异体骨/关节移植可使缺损尺寸和结构紧密匹配，同时保持活体组织的功能和愈合特性。目前，长期的免疫调节是必要的，由于免疫抑制药物或耐受诱导对这种非生命危重移植存在重大健康风险，因此不可接受。在没有免疫抑制的情况下维持同种异体组织的活力将是一项重要的进展，也是本资助续期提案的目标。治疗性血管生成用于在移植骨/关节内发展宿主来源的新血管生成循环，无论免疫状态如何，都能维持血液流动。之前的工作已经证明了这种方法的前景。本研究的具体目的是：(1)测试血管生成生长因子的局部递送是否能改善活体同种异体股骨移植中同时植入受体AV束的毛细血管形成和骨血流量；(2)确定骨形态发生蛋白的局部递送是否能在同一模型中促进新骨的形成；(3)研究新形成骨中的骨细胞是否来自受体而非移植。4)在更大的动物（兔膝）模型上评估原位全关节复合组织移植的生存能力、愈合潜力和功能。本基础研究的最终目的是提高在困难条件下进行保肢手术的临床效果。方法：在目的1、2和3中，使用隐动静脉（AV）束移植性别不匹配的带血管的股骨以提供宿主来源的血管生成。生长因子被包裹在可生物降解的微球中，放置在移植的股骨内。在两个时间点通过微血管造影定量毛细血管密度和氢冲洗法定量骨血流量来测量血管新生；而骨重塑是通过定量组织形态学荧光标记来确定的。激光捕获显微解剖将用于研究位于新形成骨区域的骨细胞谱系，通过定量实时PCR测量y染色体特异性SRY基因（从而确定细胞的宿主或移植物来源）。在兔全膝关节移植模型中，关节软骨的力学特性和关节功能的运动学分析将与血流、血管生成和组织学测量相关联，以评估该方法在复合组织异体移植中的应用。

DESCRIPTION (provided by applicant): Massive bone and joint defects arise from resection of primary and metastatic tumors, congenital deficiency, traumatic loss, infection, or prosthetic implant failure. Available reconstructive methods are prone to high rates of failure. They include structural allografts (infection, nonunion and stress fracture), vascularized autografts (limited availability, size/shape mismatch, morbidity), or prosthetic replacement (infection, periprosthetic fracture and failure). A better solution to this difficult problem is required. Transplantation of living allogenic bone/joint would allow close matching of defect dimension and structure, while simultaneously maintaining the functional and healing properties of living tissue. Long-term immune modulation is necessary at present, unacceptable due to substantial health risks of immunosuppressive drugs or tolerance induction for such non- life critical transplantation. Maintaining allogeneic tissue viability without immunosuppression would be an important advance, and is the goal of this grant renewal proposal. Therapeutic angiogenesis is used to develop a host-derived neoangiogenic circulation within the transplanted bone/joint that maintains blood flow regardless of immune status. Previous work has demonstrated the method's promise. The Specific Aims of this proposal are #1) to test whether local delivery of vasculogenic growth factors improve measures of capillary formation and bone blood flow in living femoral allotransplants with simultaneous recipient AV bundle implantation, #2) to determine whether local delivery of a bone morphogenic protein may enhance new bone formation in the same model, #3 )to investigate whether osteocytes in newly formed bone are of recipient rather than transplant origin, and #4) to evaluate the viability, healing potential and function of orthotopic whole joint composite tissue transplants in a larger animal (rabbit knee) model. Improved clinical outcome of limb salvage surgery performed in difficult circumstances is the ultimate aim of this basic research. Methods: In aims 1, 2 and 3, sex-mismatched vascularized femora are transplanted using a saphenous arteriovenous (AV) bundle to provide host-derived angiogenesis. Growth factors are delivered, encapsulated within biodegradable microspheres placed within the transplanted femur. Angiogenesis is measured at two time points by quantifying capillary density with microangiography and bone blood flow by hydrogen washout; while bone remodeling is determined after fluorochrome labeling by quantitative histomorphometry. Laser capture microdissection will be used to study lineage of osteocytes specifically located in areas of newly formed bone, as measured by quantitative real-time PCR for the Y-chromosome-specific SRY gene (thus defining host or graft origin of the cells). Following whole knee joint transplants in a rabbit model, mechanical properties of joint cartilage and kinematic analysis of joint function will be correlated with measures of blood flow, angiogenesis and histology to assess the method's use in composite tissue allotransplantation. PUBLIC HEALTH RELEVANCE: Segmental bone defects are commonly encountered in orthopedic practice but available reconstructive methods, including structural allografts, vascularized autografts and prosthetic replacement are prone to high rates of failure. Transplantation of living allogenic bone and/or joint is a potential solution that would allow close matching of defect dimension and structure, while simultaneously maintaining the functional and healing properties of living tissue. The goal of this grant renewal proposal is to study the means, mechanism of action and effectiveness of a surgically-created host-derived neoangiogenic circulation to maintain blood flow and function in living musculoskeletal tissue transplants without need for prolonged postoperative drug therapy or tolerance induction.