Novel, Rapidly Translatable Technologies for Healing Long Bone Segmental Defects

描述(由申请人提供)：本申请涉及广泛的挑战领域(11)：再生医学，以及特定的挑战主题11-AR-101肌肉骨骼和皮肤再生每年有100多万美国人因骨折住院，累计费用超过1000亿美元，如果考虑到损失工作日等因素。大型节段性骨折是临床上最具挑战性的骨折类型。例如，发生在胫骨远端的骨折通常需要多次手术才能愈合。即使成功，恢复时间也是相当长的，即使在最好的情况下，患者通常也不能在一年或更长时间内恢复完全活动。由于平民环境中高能量创伤的生存能力提高，以及伊拉克和阿富汗持续的军事冲突，这种伤害越来越常见。越来越多的大型节段性缺陷也出现在需要多次翻修失败的全关节置换术的患者中。目前治疗这种损伤的方法包括使用自体和同种异体骨移植，牵张成骨，以及应用重组人骨形态发生蛋白(BMPs)。其中每一个都有相当大的缺点。这项提案中描述的研究旨在开发一种新的修复长骨缺损的策略，该策略比现有方法更有效，成本更低，并且可以通过一次手术程序完成。它利用一种名为扩眼-冲洗器-吸引器(RIA)的新型设备，允许快速、直接和相对非侵入性地从长骨的髓内管中获取自体骨和骨祖细胞。我们之前的研究表明，与传统的骨髓抽吸法相比，放射免疫分析法回收的祖细胞更丰富，成骨能力更强。它们对重组人骨形态发生蛋白-2高度敏感，我们推测，将放射免疫分析回收的细胞和骨颗粒与重组人骨形态发生蛋白-2结合将产生强大的、协同的、成骨反应。此外，引发这种反应所需的重组人骨形态发生蛋白-2的量可能远远低于目前临床使用的量。由于重组人骨形态发生蛋白-2非常昂贵，这将极大地提高成本效益。这项提案中描述的实验将在无菌大鼠模型中对这些假设进行评估。临界大小(5 Mm)，节段性缺陷将通过手术在裸鼠的股骨中制造。这些缺陷不会自发愈合。因为这些动物是无性交的，它们会接受人类异种移植。放射免疫分析回收的人骨颗粒和骨髓细胞的组合，以及重组人骨形态发生蛋白-2将被放置到缺损处。在具体目标1中，我们将确认并扩展我们的初步发现，即当这些组件植入缺陷时，这些组件之间具有非常显著的协同作用。在具体目标2中，将优化rhBMP-2的剂量并进行时程实验，以确定BMP-2与RIA回收的材料的优化组合的骨愈合动力学。在这两个特定的目标中，每周将通过X射线监测愈合情况，直到第8周安乐死。死后，股骨将通过双能X线吸收、1/4计算机断层扫描、组织学和力学测试进行分析。具体目标3将阐述植入的人体细胞对修复缺陷的贡献。为此，RIA回收的人类骨髓细胞将在植入前用商业染料Cell Tracker Orange染色。愈合的大鼠股骨切片将被免疫组织化学染色，这些抗体识别人的核抗原，而不是大鼠的核抗原，从而对所有人的细胞核进行染色。将使用针对人类Runx2的抗体进一步探索愈合骨中任何人类细胞的身份，以识别人类成骨细胞，并使用抗人酒石酸酸性磷酸酶的抗体来识别人类破骨细胞。由于我们之前在这个项目中使用的核心技术方面的经验，该提案是“准备好的”，我们已经制定了一个积极但可行的时间表。它以季度报告要求为基础，并将允许在两年内完成工作，并明确确定的里程碑。随着这项技术进入临床应用，随着该技术应用于骨和其他结缔组织(如软骨、半月板、韧带和肌腱)的愈合过程中的其他问题，它的成功完成将产生相当大的额外的、持续的人体临床试验活动和进一步的临床前研究。我们的项目研究了一种新的修复骨折的方法，这种方法应该比现有的方法更快、更有效、更便宜。在人体试验之前，我们将在老鼠身上进行一项研究，我们将通过观察X光，通过确定骨骼有多强壮，以及通过其他方式检查骨骼，来看看是否发生了愈合。如果这个项目成功，它将避免许多人类痛苦，使人们更快地重返工作岗位，并节省资金。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (11): Regenerative Medicine, and specific Challenge Topic 11-AR-101 Musculoskeletal and Skin Regeneration Every year, over a million Americans are hospitalized for bone fractures at a cumulative cost of over $100 billion when considerations such as lost working days are factored in. Large segmental defects are the most clinically challenging types of fracture to manage. Those occurring in the distal tibia, for instance, frequently require multiple procedures to achieve union. Even when successful, the recovery time is considerable and, even under the best of circumstances, the patient often cannot return to full activity for a year or longer. Such injuries are increasingly common because of increased survivability of high energy trauma in civilian settings as well as the continuing military conflicts in Iraq and Afghanistan. Increasingly, large segmental defects are also seen in patients who have required multiple revisions of failed total joint replacements. Current approaches to treating such injuries include the use of autograft and allograft bone, distraction osteogenesis, and the application of recombinant, human bone morphogenetic proteins (BMPs). Each of these has considerable drawbacks. The research described in this proposal aims to develop a novel strategy for healing long bone defects that is more effective and far less expensive than existing methods, and can be accomplished in a single operative procedure. It utilizes a novel device known as the Reamer-Irrigator-Aspirator (RIA) that permits the rapid, straightforward and relatively non-invasive harvest of autologous bone and osteoprogenitor cells from the intramedullary canals of long bones. Our previous research has shown that the progenitor cells recovered by the RIA are more abundant and more osteogenic than marrow cells recovered by traditional bone marrow aspiration. They are highly responsive to rhBMP-2 and we hypothesize that combining the cells and osseous particles recovered by the RIA with rhBMP-2 will generate a powerful, synergistic, osteogenic response. Moreover, the amounts of rhBMP-2 needed to provoke this response are likely to be far lower than those presently used clinically. Because rhBMP-2 is so expensive, this will enormously improve cost-effectiveness. The experiments described in this proposal will evaluate these hypotheses in an athymic rat model. Critical size (5mm), segmental defects will be surgically created in the femora of athymic (nude) rats. These defects do not heal spontaneously. Because the animals are athymic, they will accept human xenografts. Combinations of human osseous particles and marrow cells recovered by the RIA, and rhBMP-2 will be placed into the defects. In Specific Aim 1, we will confirm and extend our preliminary findings of a very marked synergy between these components when implanted into the defects. In Specific Aim 2, the dose of rhBMP-2 will be optimized and time-course experiments carried out to determine the kinetics of bone healing by the optimized combination of BMP-2 and material recovered by the RIA. In these two Specific Aims, healing will be monitored by weekly X-ray until euthanasia at week 8. Post-mortem, femora will be analyzed by dual energy X- ray absorptiometry, 1/4-computed tomography, histology and mechanical testing. Specific Aim 3 will address the contributions of the implanted human cells to healing of the defect. To this end, human marrow cells recovered by the RIA will be stained with a commercial stain, Cell Tracker Orange, just prior to implantation. Sections of the healed rat femora will be stained immunohistochemically with antibodies that recognize human, but not rat, nuclear antigen and thus stain all human nuclei. The identities of any human cells within the healed bone will be further probed using antibodies against human Runx2, to identify human osteoblasts, and against human tartrate-resistant acid phosphatase, to identify human osteoclasts. Because of our prior experience with the core technologies to be used in this project, the proposal is "shovel ready" and we have developed an aggressive, but feasible, timetable. It is based upon the quarterly reporting requirements and will permit the work to be accomplished in 2 years, with clearly identified milestones. Its successful completion will generate considerable additional, sustained activity in the form of human clinical trials, as the technology moves into clinical application, and further pre-clinical research, as the technology is applied to additional problems in the healing of bone and other connective tissues, such as cartilage, meniscus, ligament and tendon. Our project investigates a new way to heal broken bones that should be quicker, more effective and less expensive than existing methods. Before trying this on people, we will undertake a study in rats and we will see whether healing has occurred by looking at X-rays, by determining how strong the bones are, and by examining the bones in other ways. If this project is successful, it will prevent much human suffering, enable people to return to work more quickly, and save money.