Enhancement of Fracture Repair by Hematopoietic Stem Cells

描述(由申请人提供)： 军事人员在战斗中骨折的风险大大增加。骨折的一个主要并发症，特别是在高能量创伤的情况下，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。骨骼重塑需要干细胞的募集和增殖，具有分化为功能成骨细胞的能力，这些细胞可以沉积和矿化细胞外的骨基质。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。利用单个增强型绿色荧光蛋白阳性(EGFP+)造血干细胞(HSC)的克隆性细胞群重建小鼠的骨髓，我们的数据表明HSC在非稳定型骨折修复过程中产生成骨细胞、骨细胞和软骨细胞。这些发现是范式的转变，因为大多数研究都集中在使用间充质干细胞修复肌肉骨骼损伤和疾病上。基于这一新的骨软骨细胞来源，我们推测HSC来源的骨软骨细胞可能被利用来促进骨折修复。建议的研究将阐明HSC的成骨细胞、骨细胞和软骨细胞分化和成熟的调控机制。在这些机制研究的基础上，HSC来源的骨软骨前体细胞将在体内进行操作，以展示它们影响骨折愈合的能力。这项研究的假设将通过两个特定的目标通过我们的新的克隆性HSC细胞移植方法结合不稳定和不愈合的骨折模型来验证。目的(1)明确HSC体外分化成熟为成骨细胞系的分子机制。本研究的目的是确定HSC向骨软骨细胞分化的机制，并确定调控HSC来源的骨软骨前体细胞分化和成熟的因素。基于分选的HSCs(Lin-SCA1+ckithiCD34-)细胞的体外实验将描述骨软骨形成谱系特异性基因的时间表达。还将分析与HSC来源的细胞分化相关的基因，以确定与分化/成熟有关的基因 来自这个独一无二的来源。互补抑制和刺激研究将被用来证明已识别基因的功能重要性。目的(2)通过体内调控HSC来源的成骨软骨前体细胞在克隆移植动物体内促进骨折修复，其中HSC来源的细胞可以追溯到单个分选的EGFP+HSC。HSC动员对骨折修复的影响将单独或结合外源性分化因子在骨折部位的应用进行检验。HSC来源的细胞对骨折愈合的贡献将通过组织化学、生物化学和显微计算机断层摄影(Micro-CT)、静态和动态组织形态计量学进行定量检测。这些研究具有重要意义，因为它们挑战了现有的教条，提出了一种新的骨和软骨细胞来源的HSC，可以用来促进骨折和骨不连的愈合。基于这种新的骨软骨干细胞来源促进和加速骨折愈合过程的方法将对军事人员具有深远的好处。考虑到高冲击性、高速度创伤，如那些在战斗中看到的，导致骨不连的风险增加，这项研究的发现与退伍军人管理局的任务有很大的相关性。拟议研究的结果有可能通过确定独特的基于干细胞的疗法来改善骨折后的康复，从而影响退伍军人健康护理。

DESCRIPTION (provided by applicant): Military personnel are at a substantially increased risk of bone fracture during combat. A major complication of fracture, especially in cases of high-energy trauma, is delayed union or non-union, meaning that the bone does not heal in a timely manner or does not heal at all. Remodeling of skeletal bone requires the recruitment and proliferation of stem cells with the capacity to differentiate to functional osteoblasts that deposit and mineralize extracellular bone matrix. Given the close association of bone and bone marrow (BM), it has been suggested that BM may serve as a source of these progenitors. Using mice whose bone marrow was reconstituted by a clonal population of cells derived from a single enhanced green fluorescent protein positive (EGFP+) hematopoietic stem cell (HSC), our data shows that the HSC gives rise to osteoblasts, osteocytes and chondrocytes during non-stabilized fracture repair. These findings are paradigm shifting in that most studies focus on the use of the mesenchymal stem cell for repair of musculoskeletal injuries and disease. Based on this novel source for osteo-chondrogenic cells, we hypothesize that HSC-derived osteo- chondrogenic cells may be exploited to enhance fracture repair. The proposed studies will elucidate mechanisms regulating differentiation and maturation of osteoblasts, osteocytes and chondrocytes from the HSC. Based on these mechanistic studies, HSC-derived osteo-chondrogenic progenitors will be manipulated in vivo to demonstrate their ability to effect healing of fracture. The hypothesis of this study will be tested using our novel clonal HSC cell transplantation method in conjunction with non-stabilized and non-union fracture models through two Specific Aims. Aim (1) is to define the molecular mechanisms regulating HSC differentiation and maturation to osteo-chondrogenic lineages in vitro. This Aim seeks to determine the mechanisms governing the commitment of HSCs to the osteo- chondrogenic lineage and identify factors regulating the differentiation and maturation of HSC-derived osteo- chondrogenic progenitors. In vitro experiments based on sorted HSCs (Lin-sca1+ckithiCD34-) cells will profile the temporal expression of osteo-chondrogenic lineage-specific genes. Genes associated with HSC-derived cell differentiation will also be profiled to identify genes involved in differentiation/maturation from this unique source. Complimentary inhibition and stimulation studies will be used to demonstrate the functional importance of identified genes. Aim (2) is to enhance fracture repair by modulation of HSC-derived osteo-chondrogenic precursors in vivo in clonally engrafted animals in which HSC-derived cells can be traced back to a single sorted EGFP+ HSC. The effects of HSC mobilization on fracture repair will be examined alone or in combination with administration of exogenous differentiation factors at the fracture site. The contribution of HSC-derived cells to fracture healing will be examined histochemically, biochemically and quantitatively using micro-computed tomography (micro-CT), static and dynamic histomorphometry. These studies are significant in that they challenge existing dogma by suggesting a novel HSC origin for bone and cartilage cells that may be exploited to enhance healing in cases of fracture and non-union. Methods to enhance and accelerate the fracture healing process based on this novel osteo-chondrogenic stem cell source would have far-reaching benefits for military personnel. Given that high-impact, high-velocity trauma such as those seen in combat, have an increased risk of resulting in non-union, the findings from this study have great relevance to the VA mission. Findings from the proposed studies have the potential to impact Veterans Health Care by identifying unique stem cell-based therapies for improving recovery from fracture.