Role of Hypoxia in Bone Formation

缺氧在骨形成中的作用

• 批准号: 7622624
• 负责人: Shawn Robert Gilbert
• 金额: $ 5.7万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7622624
• 关键词: Bone Injury; Bone Marrow; Bone and Cartilage Funding; Cartilage; Cell Differentiation process; Cell model; Cells; Chondrogenesis; Condition; Defect; Deformity; Development; Environment; Epiphysial cartilage; Fatty acid glycerol esters; Fracture; Fracture Healing; Future; Gene Expression; Goals; Growth; Healed; Histology; Hypoxia; Image; In Vitro; Injury; Lead; Marrow; Mesenchymal; Mesenchymal Differentiation; Mesenchymal Stem Cells; Mus; Muscle; Mutation; Nutrient; Osteoblasts; Osteogenesis; Oxygen; Oxygen measurement, partial pressure, arterial; Pathway interactions; Physical condensation; Physiologic calcification; Play; Polymerase Chain Reaction; Process; Proteoglycan; Role; Signal Pathway; Skeletal Development; Stromal Cells; Testing; Time; Tissues; base; bone; cartilage cell; cartilage development; desire; healing; hypoxia inducible factor 1; in vivo; in vivo Model; intramembranous bone formation; mouse model; prevent; promoter; response; single photon emission computed tomography; skeletal regeneration

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.

生长板损伤是一种独特的骨折类型，需要用软骨而不是骨愈合 以避免生长障碍和导致的畸形。间充质干细胞（MSC），驻留在 邻近骺板的骨髓间隙负责愈合损伤。MSC是多能细胞 可以根据情况分化成软骨、骨骼或脂肪组织。最近的证据表明， 局部氧利用率改变了具有低氧条件的MSC的分化，有利于软骨形成。 缺氧诱导因子1（HIF-1）是感知和响应氧气变化的关键机制。 因此，我们假设局部氧分压通过HIF-1通路改变MSC分化。 在目标1中，我们将通过确定缺氧和改变的HIF-1对MSC的影响来验证这一假设。 体外分化。将培养来自鼠骨髓的原代间充质基质细胞（MSC）， 在有利于骨或软骨分化并暴露于常氧或缺氧的条件下。分化 将通过基因表达（真实的时间PCR）和骨表型表达进行评估 （矿化）或软骨（蛋白聚糖）。类似地，来自具有条件突变的小鼠的MSC， 增加HIF-1活性（Von Hippel Lindau缺失）或降低HIF-1活性（HIF-1缺失）， 在成骨或成软骨条件下生长。为了测试HIF-1通路是否影响 当细胞向骨或软骨分化时，细胞将暴露于常氧或缺氧，并进行基因型和 检查骨或软骨标记物的表型表达。 在目标2中，我们将使用体内小鼠模型来评估MSC分化在手术创伤愈合中的作用。 在连接骨骺和干骺端骨髓腔的骺板上造成缺损， 当地的养分供应。损伤导致愈合，由膜内 骨化将通过CT和SPECT对损伤进行成像，进行详细的组织学检查，并进行基因检测。 将通过真实的时间PCR评估与缺氧、软骨形成和骨形成相关的表达 受伤的区域。 在目标3中，我们提出了开发针对MSC驱动的诱导型突变的未来方向 通过Dermol或PRXL启动子。这将允许操纵HIF-1通路（或其他期望的靶点） 以改变愈合反应，目的是防止骨 形成和由此产生的生长干扰。