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，这意味着骨头不能及时愈合或根本不愈合。骨骼骨的重塑需要具有分化为功能性成骨细胞的能力的干细胞的募集和增殖，所述功能性成骨细胞存款并矿化细胞外骨基质。鉴于骨和骨髓（BM）的密切联系，已表明BM可作为这些祖细胞的来源。使用小鼠的骨髓由来自单个增强型绿色荧光蛋白阳性（EGFP+）造血干细胞（HSC）的细胞克隆群重建，我们的数据显示，在非稳定骨折修复期间，HSC产生成骨细胞、骨细胞和软骨细胞。这些发现是范式转变，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。基于这种新的骨软骨细胞来源，我们假设HSC衍生的骨软骨细胞可能被利用来增强骨折修复。这些研究将有助于阐明HSC向成骨细胞、骨细胞和软骨细胞分化和成熟的调控机制。基于这些机制的研究，HSC衍生的骨软骨形成祖细胞将在体内被操纵，以证明其影响骨折愈合的能力。 本研究的假设将通过两个特定目的使用我们的新型克隆HSC细胞移植方法结合不稳定和不愈合骨折模型进行测试。目的：（1）明确HSC体外诱导分化成熟为骨软骨细胞的分子机制。本研究旨在确定HSC向骨软骨形成谱系定向分化的机制，并鉴定调控HSC衍生的骨软骨形成祖细胞分化和成熟的因子。基于分选的HSC（Lin-sca 1 + ckithiCD 34-）细胞的体外实验将描绘骨软骨形成谱系特异性基因的时间表达。还将分析与HSC衍生的细胞分化相关的基因以鉴定参与分化/成熟的基因 从这个独特的来源。将使用补充抑制和刺激研究来证明所鉴定基因的功能重要性。目的（2）是通过在克隆移植动物体内调节HSC衍生的骨软骨形成前体来增强骨折修复，其中HSC衍生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与骨折部位外源性分化因子的施用组合进行检查。HSC衍生细胞对骨折愈合的贡献将使用显微计算机断层扫描（micro-CT）、静态和动态组织形态计量学进行组织化学、生物化学和定量检查。 这些研究的重要性在于，它们通过提出骨和软骨细胞的新HSC来源来挑战现有的教条，这些细胞可用于增强骨折和不愈合情况下的愈合。基于这种新的骨软骨干细胞来源，增强和加速骨折愈合过程的方法将对军事人员产生深远的影响。考虑到高冲击、高速度创伤（如在战斗中所见）导致骨不连的风险增加，本研究的结果与VA使命具有很大的相关性。拟议研究的结果有可能通过确定独特的基于干细胞的治疗方法来改善骨折的恢复，从而影响退伍军人医疗保健。

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.