Fracture Repair by Mouse and Human Hematopoietic Stem Cells

描述(由申请人提供)： 摘要军事人员骨折的风险大大增加。骨折修复的一个主要并发症，特别是在高能量战斗创伤的情况下，是延迟或不愈合，这意味着骨骼不能及时愈合或根本不愈合。骨修复需要招募具有分化为矿化功能的成骨细胞的干细胞。鉴于骨和骨髓(BM)之间的密切联系，有人认为BM可能是这些祖细胞的来源之一。初步的体内研究表明，由单个增强型绿色荧光蛋白阳性(EGFP+)造血干细胞(HSC)来源的克隆群体的小鼠骨髓重建的EGFP+细胞具有成骨细胞、骨细胞和软骨细胞的形态特征。数据还显示，在非稳定型骨折修复过程中，这些细胞增加。这些发现支持我们的假设，即成骨细胞、骨细胞和软骨细胞起源于造血干细胞。我们建议阐明HSCs在骨折愈合中的作用，鉴定具有这种成骨软骨形成潜力的人类干细胞，并利用这种独特的骨祖细胞来源来促进骨折愈合。这一假说将通过以下两个特定的目的通过以下两个特定目的进行验证：1)研究HSC在骨折修复过程中对成骨软骨细胞谱系的作用。这一目标将利用我们建立的小鼠移植模型，结合稳定的、不稳定的骨折和不愈合的骨折，从组织化学、生化和形态计量学角度研究骨折骨痂中的HSC来源的细胞类型。这一目的还将检验单独应用粒细胞集落刺激因子(G-CSF)或联合应用骨形态发生蛋白(BMP)在骨折部位动员HSC的效果。2)研究人干细胞在骨折修复过程中对成骨细胞系的作用。基于表面抗原和染料排斥的FACS分选将用于鉴定和丰富参与异种移植模型中骨折修复的人类干细胞。还将检查G-CSF增加其参与的能力。这些研究具有重要意义，因为它们提出了骨折修复过程中骨和软骨细胞的一种新的HSC来源。在动员这种独特的骨软骨性HSC来源的基础上，加强和加速骨折愈合的方法将对军事人员产生深远的好处。鉴于高冲击性创伤增加了形成骨不连的风险，这些发现与退伍军人管理局的任务有很大的相关性，并有可能通过确定改善骨折恢复的独特目标来影响退伍军人保健。 公共卫生相关性： 与此相关的是，军事人员骨折的风险大大增加，特别是那些由高能量战斗相关创伤造成的骨折。在这种情况下，骨折修复的一个主要并发症是延迟愈合或不愈合，这意味着骨骼不能及时愈合或根本不愈合。骨骼的修复需要干细胞的募集和增殖，并具有分化为功能性骨细胞的能力。我们的研究表明，造血干细胞(HSC)是这种修复过程的一种新的干细胞来源。我们的数据还表明，在非稳定型骨折修复过程中，这些细胞增加。这些研究的目的是阐明HSCs在骨折愈合中的作用，鉴定具有这种成软骨潜能的人类干细胞，并利用这种独特的骨祖细胞来源来促进骨折愈合。在动员这种特殊的骨软骨干细胞来源的基础上，加强和加速骨折愈合过程的方法将对军事人员产生深远的好处。缩短恢复完全机动和功能范围的时间将减少医疗费用，缩短新兵在基本训练中受伤后进入现役的时间，并加速职业士兵重返战斗。此外，加强愈合过程可能会通过减轻疼痛、增加活动能力和防止骨折不愈合导致的长期残疾来提高生活质量。

DESCRIPTION (provided by applicant): ABSTRACT Military personnel are at a substantially increased risk of bone fracture. A major complication of fracture repair, especially in cases of high-energy combat trauma, is delayed or non-union, meaning that bone does not heal in a timely manner or does not heal at all. Bone repair requires the recruitment of stem cells with the capacity to differentiate to functional osteoblasts that mineralize. Given the close association of bone and bone marrow (BM), it has been suggested that BM may serve as a source of these progenitors. Preliminary in vivo studies of mice whose BM was reconstituted by a clonal population of cells derived from a single enhanced green fluorescent protein positive (EGFP+) hematopoietic stem cell (HSC) show EGFP+ cells with morphological characteristics of osteoblasts, osteocytes, and chondrocytes. Data also demonstrate an increase in these cells during non-stabilized fracture repair. These findings support our hypothesis that osteoblasts, osteocytes, and chondrocytes are derived from HSCs. We propose to elucidate the contribution of HSCs to fracture healing, identify the human stem cell that possess this oste-chondrogenic potential, and exploit this unique source of osteoprogenitor cells to augment fracture healing. This hypothesis will be tested using both clonal cell mouse-to-mouse and xenograft transplantation methods in conjunction with stabilized, non-stabilized and non-union fracture models through two Specific Aims: 1) To examine the temporal and functional contribution of HSCs to oste-chondrogenic lineages during fracture repair. This Aim will utilize our established murine transplantation model in conjunction with stabilized, non-stabilized fracture and non-union fractures to histochemically, biochemically and morphometrically examine the HSC-derived cell types in the fracture callus. This aim will also examine the effects of HSC mobilization via granulocyte-colony stimulating factor (G-CSF) administration alone or in combination with administration of bone morphogenic protein (BMP) at the fracture site. 2) To examine the contribution of human stem cells to osteo-chondrogenic lineages during fracture repair. FACS-sorting based on surface antigens and dye exclusion will be used to identify and enrich for human stem cells that participate in fracture repair in a xenograft model. The ability of G-CSF to augment their participation will also be examined. These studies are significant in that they suggest a novel HSC origin for bone and cartilage cells during fracture repair. Methods to enhance and accelerate fracture healing based on mobilization of this unique osteo- chondrogenic HSC source would have far-reachingbenefits for military personnel. Given that high-impact trauma have increased risk of forming non-union, these findings have great relevance to the VA mission and have potential to impact Veterans Health Care by identifying unique targets to improve fracture recovery. PUBLIC HEALTH RELEVANCE: RELEVANCE Military personnel are at a substantially increased risk of bone fracture, especially those resulting from high- energy combat-related trauma. A major complication of fracture repair in such cases is delayed union or non- union, meaning that the bone does not heal in a timely manner or does not heal at all. Repair of skeletal bone requires the recruitment and proliferation of stem cells with the capacity to differentiate to functional bone cells. Our studies demonstrate that the hematopoietic stem cell (HSC) is a novel source of stem cells for this repair process. Our data also demonstrate an increase in these cells during non-stabilized fracture repair. The goal of these studies is to elucidate the contribution of HSCs to fracture healing, identify the human stem cell that possess this oste-chondrogenic potential, and exploit this unique source of osteoprogenitor cells to augment fracture healing. Methods to enhance and accelerate the fracture healing process based on mobilization of this particular osteo-chondrogenic stem cell source would have far-reachingbenefits for military personnel. Decreasingthe time to return to complete mobility and range of function would reduce medicalcosts, decrease the time for military recruits toenter active duty after injuries incurred in basic training, and accelerate the return of professional soldiers to combat. Moreover, augmenting the healing process may enhance quality of life by decreasing pain, increasingmobility and preventing the long-termdisability caused by fracture non- union.