Role of Angiogenesis in Distraction Osteogenesis

血管生成在牵张成骨中的作用

• 批准号: 8321521
• 负责人: Louis Charles Gerstenfeld
• 金额: $ 36.83万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8321521
• 关键词: Ablation; Address; Antibodies; BMP2 gene; Back; Belief; Blood Vessels; Bone Regeneration; Bone Tissue; Cartilage; Cell Differentiation process; Cell Line; Cells; Clinical; Coculture Techniques; Commit; Congenital Abnormality; Data; Development; Distraction Osteogenesis; Endothelial Cells; Endothelium; Excision; Extracellular Matrix; Feedback; Fracture; Galactosidase; Gene Targeting; Genes; Genetic Recombination; Growth Factor; Healed; Impaired wound healing; Injury; Laboratories; Lead; Malignant Neoplasms; Mediating; Mesenchymal Stem Cells; Modeling; Molecular; Morbidity - disease rate; Morphogenesis; Mus; Natural regeneration; Operative Surgical Procedures; Orthopedics; Osteoblasts; Osteogenesis; Osteotomy; Phase; Play; Population; Procedures; Process; Production; Proteins; Recruitment Activity; Relative (related person); Role; Site; Source; Testing; Tissues; Transgenes; Transgenic Mice; Trauma; Vascular Endothelial Growth Factor Receptor; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factors; Vascular Endothelium; Vascular blood supply; Work; angiogenesis; bone; bone healing; bone morphogenetic protein 2; cell growth; distraction; functional status; healing; innovation; osteogenic; paracrine; receptor-mediated signaling; regenerative; repaired; research study; selective expression; skeletal; skeletal injury; small hairpin RNA; vasculogenesis

DESCRIPTION (provided by applicant): Compromised healing of skeletal injuries is associated with increased morbidity and decreased functional status, and each year surgical orthopedic treatments are performed on over 800,000 fractures with impaired healing. Inadequate blood supply is a major cause of impaired healing in all types of orthopedic treatments. Bone morphogenetic protein 2 (BMP2) plays an essential role in promoting mesenchymal stem cell (MSC) osteogenic differentiation; while vascular endothelia growth factors (VEGFs) play crucial roles in the recruitment and differentiation of vascular endothelia cells (ECs). Currently, it is commonly accepted that BMP2 is either produced by MSCs and osteogenic cells or is proteolytically released from sequestered stores in the extracellular matrix. Our preliminary studies with transgenic mice have shown that ECs that line vessels adjacent to both fracture and osteotomy sites, and within bone repair tissues, are the main source of BMP2 at the repair site. VEGFs have also been shown to function in bone healing however, whether they act in a direct manner via receptor-mediated signaling in osteogenic cells is highly debated. Other preliminary work from our laboratory has shown that while differentiated osteoblasts abundantly express VEGF(s), exogenously added VEGF(s) neither promote MSC osteogenic differentiation nor enhance differentiated osteoblast function. Collectively, these data lead us to the following hypothesis: The development of vascular and bone tissues during post natal bone repair and regeneration is regulated through a positive paracrine feedback loop, which is established through the expression of BMP2 by ECs and VEGF expression by osteogenic cells. This hypothesis is a paradigm shift from prior general beliefs about the cell sources and functions of BMP2 and VEGF during bone repair. A murine model of distraction osteogenesis (DO) will be used to address this hypothesis. DO has three advantages for the proposed studies: a) new bone formation occurs primarily through an intramembranous process devoid of extensive cartilage; b) it is an extremely robust regenerative model capable of inducing immense amounts of highly vascularized bone tissues; c) it is successfully used in the clinical setting to treat some forms of nonunions in the absence of BMP2 treatment. Aim 1 will define the functional relationship between vessel morphogenesis and the induction of BMP2 in endothelial cells by altering vascular morphogenesis through blocking VEGFR 1 and 2 activities. Using transgenic mice containing a conditionally inducible Cre gene that is selectively expressed in endothelial cells, the development of nascent endothelial cells will be tracked within new vessels and co-localized to the cell populations that are expressing BMP2. Aim 2 will use these same transgenic mice to assess the functional role of BMP2 expression by endothelial cells in the formation of bone and vascular tissues by BMP2 gene ablation in ECs during the different phases of DO. The completion of Aims 1 and 2 will determine if ECs are a primary cell source of BMP2 in regulating skeletal healing after surgery or injury and if BMP2 has a functional role in vasculogenesis.

描述（由申请人提供）：骨骼损伤愈合受损与发病率增加和功能状态下降相关，每年对超过 800,000 起愈合受损的骨折进行外科骨科治疗。血液供应不足是所有类型的骨科治疗中愈合受损的主要原因。骨形态发生蛋白2（BMP2）在促进间充质干细胞（MSC）成骨分化中发挥重要作用；而血管内皮生长因子（VEGF）在血管内皮细胞（EC）的招募和分化中发挥着至关重要的作用。目前，人们普遍认为 BMP2 要么由 MSC 和成骨细胞产生，要么从细胞外基质中的隔离储存中通过蛋白水解释放。我们对转基因小鼠的初步研究表明，位于骨折和截骨部位附近以及骨修复组织内的血管内皮细胞是修复部位 BMP2 的主要来源。 VEGF 也已被证明在骨愈合中发挥作用，然而，它们是否通过成骨细胞中受体介导的信号传导直接发挥作用仍存在很大争议。我们实验室的其他前期工作表明，虽然分化的成骨细胞大量表达VEGF，但外源添加的VEGF既不能促进MSC成骨分化，也不能增强分化的成骨细胞功能。总的来说，这些数据使我们得出以下假设：出生后骨修复和再生过程中血管和骨组织的发育是通过正旁分泌反馈回路调节的，该反馈回路是通过 EC 表达 BMP2 和成骨细胞表达 VEGF 建立的。这一假设是对之前关于骨修复过程中 BMP2 和 VEGF 的细胞来源和功能的普遍看法的范式转变。小鼠牵张成骨（DO）模型将用于解决这一假设。对于拟议的研究来说，DO 具有三个优点：a）新骨形成主要通过缺乏广泛软骨的膜内过程发生； b) 它是一种极其强大的再生模型，能够诱导大量高度血管化的骨组织； c) 它已成功应用于临床，在没有 BMP2 治疗的情况下治疗某些形式的骨不连。目标 1 将通过阻断 VEGFR 1 和 2 活性来改变血管形态发生，从而定义血管形态发生与内皮细胞中 BMP2 诱导之间的功能关系。使用含有在内皮细胞中选择性表达的条件诱导Cre基因的转基因小鼠，新生内皮细胞的发育将在新血管内被追踪，并与表达BMP2的细胞群共定位。目标 2 将使用这些相同的转基因小鼠，通过在 DO 的不同阶段消除 EC 中的 BMP2 基因，评估内皮细胞表达的 BMP2 在骨和血管组织形成中的功能作用。目标 1 和 2 的完成将确定 EC 是否是 BMP2 在调节手术或损伤后骨骼愈合中的主要细胞来源，以及 BMP2 是否在血管生成中具有功能性作用。