Role of Hypoxia in Bone Formation

生长板损伤是一种独特的骨折类型，需要用软骨而不是骨骼来愈合。 以避免生长障碍和由此导致的畸形。间充质干细胞(MSCs)位于 骨赘附近的骨髓间隙负责愈合损伤。间充质干细胞是一种多潜能细胞 可以根据情况分化为软骨、骨或脂肪组织。最近的证据表明， 局部氧气供应改变了低氧条件下MSCs的分化，有利于软骨形成。 缺氧诱导因子-1(HIF-1)是感知和响应氧变化的关键机制。 因此，我们假设局部氧分压通过HIF-1途径改变MSC分化。 在目标1中，我们将通过确定低氧和改变HIF-1对MSC的影响来检验这一假说 体外分化。小鼠骨髓间充质基质细胞(MSCs)将被培养 在有利于骨骼或软骨分化的条件下，暴露在常氧或低氧环境中。差异化 将通过基因表达(实时聚合酶链式反应)和骨的表型表达来评估 (矿化)或软骨(蛋白多糖)。同样，携带条件性突变的小鼠的MSCs 增加HIF-1活性(冯·希佩尔·林道缺失)或降低HIF-1活性(HIF-1缺失) 生长在成骨或成软骨的条件下。为了测试HIF-1通路是否影响了 分化为骨或软骨时，细胞将暴露在常氧或低氧环境中，并表现为 将检查骨或软骨标志物的表型表达。 在目标2中，我们将使用活体小鼠模型来评估骨髓间充质干细胞分化在手术愈合中的作用。 在连接骨骺和干骺端的骨痂上造成了缺陷，改变了 当地的养分可利用性。损伤导致愈合，由膜内形成的骨桥 骨化。损伤将通过CT和SPECT进行成像，将进行详细的组织学检查，并对 实时定量聚合酶链式反应将评估与缺氧、软骨生成和成骨相关的表达。 受伤区域的位置。 在目标3中，我们提出了针对MSC驱动的可诱导突变的未来发展方向 通过皮下或PRXL启动子。这将允许操纵HIF-1途径(或其他所需的靶点) 在骨髓间充质干细胞分化之前，为了改变愈合反应，目的是防止骨骼 形成和由此产生的生长障碍。

Growth plate injuries are a unique type of fracture where healing with cartilage instead of bone is desirable to avoid growth disturbance and resulting deformity. Mesenchymal stem cells (MSCs) that reside in the marrow spaces adjacent to the physis are responsible for healing the injuries. MSCs are pluriopotent cells that can differentiate to cartilage, bone or fat tissue based on conditions. Recent evidence has shown that local oxygen availability alters the differentiation of MSCs with hypoxic conditions favoring chondrogenesis. Hypoxia Inducible Factor 1 (HIF-1) is a key mechanism for sensing and responding to changes in oxygen. Therefore, we hypothesize that local oxygen tension alters MSC differentiation via the HIF-1 pathway. In Aim 1, we will test this hypothesis by determining the effects of hypoxia and altered HIF-1 on MSC differentiation in vitro. Primary mesenchymal stromal cells (MSCs) from murine bone marrow will be grown in conditions favoring bone or cartilage differentiation and exposed to normoxia or hypoxia. Differentiation will be assessed by gene expression (real time PCR) and by phenotypic expression of bone (mineralization) or cartilage (proteoglycans). Similarly, MSCs from mice with conditional mutations to increase HIF-1 activity (Von Hippel Lindau deletion) or decrease HIF-1 activity (HIF-1 deletion) will then be grown in osteogenic or chondrogenic conditions. To test whether the HIF-1 pathway impinges on differentiation to bone or cartilage, the cells will be exposed to normoxia or hypoxia and genotypic and phenotypic expression of bone or cartilage markers will be examined. In Aim 2, we will use an in vivo mouse model to evaluate MSC differentiation in healing of a surgically created defect across the physis that connects the epiphyseal and metaphyseal marrow spaces, altering local nutrient availability. The injury results in healing with a bony bridge formed by intramembranous ossification. Injuries will be imaged by CT and SPECT, detailed histology will be performed, and gene expression associated with hypoxia, chondrogenesis, and osteogenesis will be evaluated by real time PCR of the zone of injury. In Aim 3, we propose a future direction for development of an inducible mutation targeted to MSC's driven by the dermol or prxl promoter. This will allow manipulation of the HIF-1 pathway (or other desired target) in MSC's prior to differentiation in order to alter the healing response with the goal of preventing bone formation and the resulting growth disturbance.