Rapid mobilization of endogenous progenitor cells for bone healing

描述（由申请人提供）：愈合受损和不愈合是骨折的严重并发症，导致疼痛、畸形、功能和活动能力下降。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，建立痂，最终使骨恢复到原来的形状和机械完整性。骨髓干细胞和祖细胞移植到损伤部位是一种有效的促进愈合的治疗方法。骨髓是内皮祖细胞（EPCs）、间充质干细胞（MSCs）和造血干细胞（hsc）的丰富来源，它们已被证明可以诱导血管生成和骨生成，并释放抗炎细胞因子以促进骨愈合。然而，诸如此类的基于细胞的治疗方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迅速动员大量内源性祖细胞直接进入外周血（PB），这些细胞将返回损伤部位并参与组织再生。AMD3100是趋化因子（CXC motif）受体4 （CXCR4）的选择性拮抗剂，可快速动员hsc和EPCs进入PB。AMD3100联合VEGF可动员MSCs。我们的中心假设是，骨折后骨髓来源的祖细胞被释放到PB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测定和流式细胞术定量小鼠骨折和AMD3100/VEGF时动员的MSCs、hsc和EPCs；B)通过追踪被动员的祖细胞到骨折部位的归巢；C)衡量效果

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.