Rapid mobilization of endogenous progenitor cells for bone healing

描述(申请人提供)：愈合受损和骨不连是骨折的严重并发症，会导致疼痛、畸形、功能减退和行动不便。骨愈合是一个复杂的过程，需要干细胞和祖细胞迁移来修复血管系统，建立骨痂，最终使骨骼恢复到原来的形状和机械完整性。将骨髓来源的干细胞和祖细胞移植到损伤部位是促进愈合的有效治疗方法。骨髓中含有丰富的内皮祖细胞(EPC)、间充质干细胞(MSCs)和造血干细胞(HSCs)，它们可以诱导血管生成和成骨，并释放抗炎细胞因子促进骨愈合。然而，这类基于细胞的疗法需要分离骨髓，并在体外扩增或浓缩细胞。一种完全不同的方法是快速动员大量内源性祖细胞直接进入外周血液(PB)，外周血液将成为损伤部位的归宿，并参与组织再生。AMD3100是趋化因子(CXC基序)受体4(CXCR4)的选择性拮抗剂，能迅速动员HSCs和EPC进入外周血。AMD3100联合血管内皮生长因子动员间充质干细胞。我们的中心假设是，骨折后，骨髓来源的祖细胞被释放到外周血中，通过药物干预增加这些细胞的循环数量将促进骨愈合。我们将验证这一假设a)通过使用集落形成单位分析和流式细胞术来量化骨折后动员的MSCs、HSCs和EPC以及AMD3100/VEGF；b)通过跟踪动员的祖细胞到骨折部位的归巢；c)通过测量 使用小鼠骨折模型研究血管内皮生长因子/AMD3100对骨愈合的影响。我们建议在骨折愈合的前三天动员细胞，此时炎症细胞因子和化学吸引分子被释放，细胞被认为是损伤部位的家。我们预计骨折会导致外周血中MSCs、HPC和EPC数量的增加，药物干预可以进一步显著增加这些数量。我们预计早期，更多的循环祖细胞可用于骨折部位和 通过植入、细胞分化和组织细化直接促进组织愈合，通过释放旁分泌因子间接促进骨愈合。更大的骨痂大小、组织矿物质密度和生物力学强度将证明促进愈合。我们的总体目标是证明将内源性骨髓前体细胞快速释放到外周血中是促进组织再生的有效策略。预计在损伤后不久就能有效地动员干细胞，可以避免骨髓抽吸的需要，以及与体外细胞操作相关的并发症。

DESCRIPTION (provided by applicant): Impaired healing and non-union are a serious complication of a fracture that results in pain, deformity, diminished function and mobility. Bone healing is a complex process which requires stem and progenitor cell migration to repair the vasculature, establish a callus and ultimately return the bone to its former shape and mechanical integrity. Delivery of bone marrow derived stem and progenitor cells to the site of injury is an effective therapy to enhance healing. Bone marrow is a rich source of endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs), which have been shown to induce angiogenesis and osteogenesis and release anti-inflammatory cytokines to enhance bone healing. However, cell-based therapeutics such as these require the isolation of bone marrow and expansion or concentration of cells in vitro. A radically different approach is to rapidly mobilize large numbers of endogenous progenitor cells directly into the peripheral blood (PB) that will home to the site of injury and take part in tissu regeneration. AMD3100, is a selective antagonist of chemokine (CXC motif) receptor 4 (CXCR4) that rapidly mobilizes HSCs and EPCs into PB. AMD3100 in combination with VEGF mobilizes MSCs. Our central hypothesis is that bone marrow-derived progenitor cells are released into the PB subsequent to fracture, and that increasing circulating numbers of these cells using pharmacological interventions will improve bone healing. We will test this hypothesis a) by quantifying MSCs, HSCs and EPCs mobilized in response to fracture and AMD3100/VEGF in the mouse using colony forming unit assays and flow cytometry; b) by tracking the homing of mobilized progenitor cells to the fracture site; c) by measuring the effects of VEGF/AMD3100 on bone healing using a murine fracture model. We propose to mobilize cells during the first three days of fracture healing, at a time when inflammatory cytokines and chemoattractant molecules are released, and cells are thought to home to the site of injury. We anticipate that fracture results in increased numbers of MSCs, HPCs and EPCs in PB and that pharmacological intervention can significantly increase these numbers further. We anticipate that early, increased availability of circulating progenitor cells to home to the fracture site and contribute directly to tissue healing by engraftment, cell differentiation and tissue elaboration o indirectly through release of paracrine factors will accelerate bone healing. Enhanced healing will be evidenced by a greater callus size, tissue mineral density, and biomechanical strength. Our overall objective is to show that rapid release of endogenous bone marrow progenitors into PB is an effective strategy to enhance tissue regeneration. It is expected that efficient mobilization of stem cells shortly after injury, could circumvent the need for bone marrow aspiration, and complications associated with in vitro cell manipulation.