Novel, Rapidly Translatable Technologies for Healing Long Bone Segmental Defects

用于治疗长骨段缺损的新颖、快速可转化技术

• 批准号: 7943919
• 负责人: CHRISTOPHER Howard EVANS
• 金额: $ 48.02万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7943919
• 关键词: Address; Afghanistan; American; Animals; Antibodies; Area; Arthroplasty; Aspirate substance; Autologous; Autologous Transplantation; Biological; Bone Marrow Aspiration; Bone Marrow Cells; Bone Morphogenetic Proteins; Cartilage; Cell Culture Techniques; Cell Nucleus; Cells; Clinical; Clinical Research; Clinical Trials; Conflict (Psychology); Connective Tissue; Defect; Deposition; Devices; Distal; Distraction Osteogenesis; Dose; Dual-Energy X-Ray Absorptiometry; Economics; Embolism; Euthanasia; Femur; Filler; Fracture; Harvest; Healed; Hematopoietic; Hip region structure; Histology; Human; Immunohistochemistry; Implant; Incidence; Injury; Iraq; Kinetics; Label; Laboratories; Ligaments; Marrow; Mechanics; Meniscus structure of joint; Methods; Military Personnel; Modeling; Monitor; Morbidity - disease rate; Musculoskeletal; Nuclear Antigens; Nude Rats; Occupations; Operative Surgical Procedures; Oranges; Orthopedics; Osteoblasts; Osteoclasts; Patients; Population; Procedures; Protocols documentation; Rattus; Recombinants; Recovery; Regenerative Medicine; Replacement Arthroplasty; Reporting; Research; Roentgen Rays; Site; Source; Staining method; Stains; Stem cells; Study models; Surface; Surgeon; Technology; Tendon structure; Testing; Time; Translations; Trauma; Vertebral column; Work; X-Ray Computed Tomography; Xenograft procedure; allogenic bone transplantation; base; bone; bone healing; bone morphogenetic protein 2; bone quality; clinical application; clinical practice; cost; cost effectiveness; design; experience; healing; human data; human tissue; implantation; improved; long bone; novel; novel strategies; osteogenic; osteoprogenitor cell; particle; pre-clinical research; precursor cell; pressure; prevent; recombinant human bone morphogenetic protein-2; research study; response; skin regeneration; tartrate-resistant acid phosphatase; tibia; tissue regeneration

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.

描述（由申请人提供）：本申请涉及广泛的挑战领域 (11)：再生医学和具体挑战主题 11-AR-101 肌肉骨骼和皮肤再生 每年，超过一百万美国人因骨折住院，累计费用为如果考虑到工作日损失等因素，这一损失将超过 1000 亿美元。大节段性缺损是临床上最具挑战性的骨折类型。例如，发生在胫骨远端的那些，经常需要多次手术才能实现愈合。即使成功，恢复时间也很长，而且即使在最好的情况下，患者也常常在一年或更长时间内无法完全恢复活动。由于平民环境中高能量创伤的生存能力以及伊拉克和阿富汗持续的军事冲突，此类伤害越来越常见。在需要对失败的全关节置换术进行多次翻修的患者中，越来越多地出现大的节段缺损。目前治疗此类损伤的方法包括使用自体移植骨和同种异体移植骨、牵引成骨以及应用重组人骨形态发生蛋白（BMP）。其中每一个都有相当大的缺点。该提案中描述的研究旨在开发一种治疗长骨缺损的新策略，该策略比现有方法更有效且成本低得多，并且可以在单次手术中完成。它利用一种称为扩孔器-冲洗器-吸引器 (RIA) 的新型装置，可以快速、直接且相对无创地从长骨髓内管中获取自体骨和骨祖细胞。我们之前的研究表明，通过 RIA 回收的祖细胞比通过传统骨髓抽吸回收的骨髓细胞更丰富且更具成骨性。它们对 rhBMP-2 高度敏感，我们假设将 RIA 回收的细胞和骨颗粒与 rhBMP-2 结合将产生强大的协同成骨反应。此外，引发这种反应所需的 rhBMP-2 量可能远低于目前临床使用的量。由于rhBMP-2非常昂贵，这将极大地提高成本效益。本提案中描述的实验将在无胸腺大鼠模型中评估这些假设。将通过手术在无胸腺（裸鼠）大鼠的股骨中产生临界尺寸（5毫米）的节段性缺损。这些缺陷不会自行愈合。因为这些动物没有胸腺，所以它们会接受人类异种移植物。通过 RIA 回收的人类骨颗粒和骨髓细胞以及 rhBMP-2 的组合将被放入缺损处。在具体目标 1 中，我们将确认并扩展我们的初步发现，即这些组件在植入缺陷时会产生非常显着的协同作用。在具体目标 2 中，将优化 rhBMP-2 的剂量并进行时程实验，以确定通过 BMP-2 和 RIA 回收的材料的优化组合来确定骨愈合的动力学。在这两个具体目标中，将通过每周 X 射线监测愈合情况，直至第 8 周安乐死。尸检时，将通过双能 X 射线吸收测定法、1/4 计算机断层扫描、组织学和机械测试对股骨进行分析。具体目标 3 将解决植入的人体细胞对修复缺陷的贡献。为此，在植入之前，通过 RIA 回收的人类骨髓细胞将用商业染色剂 Cell Tracker Orange 进行染色。愈合的大鼠股骨切片将用识别人类核抗原但不识别大鼠核抗原的抗体进行免疫组织化学染色，从而对所有人类细胞核进行染色。将使用抗人 Runx2 的抗体进一步探测愈合骨内任何人类细胞的身份，以识别人成骨细胞，并使用抗人抗酒石酸酸性磷酸酶的抗体，以识别人破骨细胞。由于我们之前对该项目中使用的核心技术的经验，该提案已“准备就绪”，并且我们制定了一个积极但可行的时间表。它基于季度报告要求，并将允许在两年内完成工作，并明确确定里程碑。随着该技术进入临床应用，它的成功完成将以人体临床试验的形式产生大量额外的、持续的活动，以及进一步的临床前研究，因为该技术应用于骨和其他结缔组织愈合中的其他问题，如软骨、半月板、韧带和肌腱。我们的项目研究了一种治疗骨折的新方法，该方法比现有方法更快、更有效且更便宜。在对人类进行尝试之前，我们将在老鼠身上进行一项研究，我们将通过观察 X 射线、确定骨骼的强度以及通过其他方式检查骨骼来了解是否已发生愈合。如果该项目成功，将避免许多人类痛苦，使人们能够更快地重返工作岗位，并节省资金。

项目成果

Interaction between living bone particles and rhBMP-2 in large segmental defect healing in the rat femur.

活骨颗粒和 rhBMP-2 在大鼠股骨大节段缺损愈合中的相互作用。

• 发表时间: 2016
• 作者: Liu, Fangjun;Wells, James W;Porter, Ryan M;Glatt, Vaida;Shen, Zhenxin;Schinhan, Martina;Ivkovic, Alan;Vrahas, Mark S;Evans, Christopher H;Ferreira, Elisabeth
• 通讯作者: Ferreira, Elisabeth