Angiogenesis and Long-term Bone and Joint Allotransplant Survival

描述(申请人提供)：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引起的大量骨和关节缺陷。现有的重建方法容易出现高失败率。它们包括结构异体移植(感染、骨不连和应力性骨折)、带血管自体移植(可用性有限、大小/形状不匹配、发病率)或假体置换(感染、假体周围骨折和失败)。这一难题需要一个更好的解决方案。活体同种异体骨/关节移植将使缺损区的大小和结构紧密匹配，同时保持活组织的功能和愈合特性。目前，长期的免疫调节是必要的，但由于免疫抑制药物的巨大健康风险或此类非生命关键移植的耐受诱导，这是不可接受的。在没有免疫抑制的情况下维持同种异体组织的活性将是一项重要的进步，也是这项赠款续期提案的目标。治疗性血管生成用于在移植的骨/关节内形成宿主来源的新生血管循环，无论免疫状态如何，该循环都能维持血液流动。以前的工作已经证明了该方法的前景。该建议的具体目的是：1)测试局部输送血管生成生长因子是否能改善同种异体股骨移植中毛细血管形成和骨血流的测量；2)确定局部输送骨形态发生蛋白是否可以在相同的模型中促进新骨形成；#3)研究新形成的骨中的骨细胞是否来自受体，以及#4)在更大的动物(兔膝关节)模型中评估原位全关节复合组织移植的存活率、愈合潜力和功能。改善在困难环境下进行的保肢手术的临床结果是这项基础研究的最终目的。方法：在AIMS 1、2和3中，使用隐动静脉束移植性别不匹配的带血管股骨，以提供宿主来源的血管生成。生长因子被输送，被封装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血管密度和氢气冲洗法测量骨血流量来测量血管生成；而荧光标记后通过定量组织形态计量学测量骨改建。激光捕获显微切割将被用来研究特定位于新骨形成区域的骨细胞谱系，通过Y染色体特异的SRY基因的实时定量聚合酶链式反应(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量、血管生成和组织学指标相关联，以评估该方法在同种异体复合组织移植中的应用。 公共卫生相关性：节段性骨缺损在骨科实践中很常见，但现有的重建方法，包括结构同种异体骨移植、带血管自体骨移植和假体置换，容易出现高失败率。活体同种异体骨和/或关节移植是一种潜在的解决方案，它可以在保持活组织的功能和愈合特性的同时，实现缺损区尺寸和结构的紧密匹配。这项拨款更新提案的目的是研究通过手术创造的宿主来源的新生血管循环的手段、作用机制和有效性，以维持活体肌肉骨骼组织移植的血液流动和功能，而不需要长期的术后药物治疗或耐受诱导。

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.