Defining the Tendon Lineage to Improve Tissue Engineering Strategies

定义肌腱谱系以改进组织工程策略

• 批准号: 9105160
• 负责人: Nathaniel A. Dyment
• 金额: $ 10.8万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-02 至 2017-01-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9105160
• 关键词: Address; Adult; Affect; Back; Biological; Biological Models; Biology; Biomechanics; Bromodeoxyuridine; Cell Cycle; Cell surface; Cells; Connecticut; Data; Defect; Development; Differentiation and Growth; Embryonic Development; Engineering; Environment; Epitopes; Fibroblasts; Frequencies; Future; Goals; Growth; Healed; Health; Heterogeneity; Human; Immunodeficient Mouse; Injury; Knock-out; Knowledge; Label; Lead; Literature; Maps; Measures; Mechanics; Mediating; Mentors; Methods; Microscopy; Modeling; Molecular; Molecular Profiling; Mus; Musculoskeletal; Mutant Strains Mice; Normal Cell; Operative Surgical Procedures; Outcome; Pathway interactions; Patients; Phase; Population; Process; Property; Proteomics; Quality of life; Reporter; Research; Research Personnel; Research Training; Signal Pathway; Signal Transduction; Smooth Muscle Actin Staining Method; Source; Specific qualifier value; Staging; Stem cells; Tendon Injuries; Tendon structure; Testing; Therapeutic; Tissue Engineering; Tissues; Training; Transforming Growth Factor beta; Transgenic Organisms; Universities; Work; analog; base; career; cell type; differential expression; experience; healing; improved; in vivo; in vivo Model; injury and repair; insight; ligament injury; mouse model; musculoskeletal injury; novel; postnatal; prevent; progenitor; promoter; public health relevance; receptor; repaired; scleraxis; skeletal; skills; stem; success; tendon development; therapy development; tool; transcriptome; two-photon

 DESCRIPTION (provided by applicant): My career goal is to become an independent investigator that contributes to the understanding of tendon biology and develops improved strategies for tendon repair. My research training started as an engineer who focused on developing therapies that return tendon back to normal mechanical function. I realized that an incomplete knowledge of the cellular mechanisms that lead to improved repair was preventing me from developing an effective therapeutic strategy. I decided that I needed to improve my understanding of the origin of tendon progenitors, the markers that define their differentiation state, and the signaling pathways that regulate their differentiation prior to developing novel repair strategies. My mentoring team, consisting of my primary mentor Dr. David Rowe and other skilled musculoskeletal biologists and engineers at the University of Connecticut Health Center along with an outside tendon developmental biologist, provide me with an exceptional environment to investigate these questions and develop the necessary skills to contribute to the field as an independent investigator. We have developed a repertoire of transgenic lineage tracing and GFP reporter mice that identifies resident tendon progenitor populations and showcases the heterogeneity of cells in the tendon midsubstance that was once considered quite homogeneous. This application lays out a strategy to characterize the transciptomic and proteomic profile of resident tendon progenitors as they differentiate during normal processes of tendon growth and natural healing following injury. The central hypothesis is that the molecular mechanisms that drive tendon progenitor differentiation during growth are crucial to improving tendon repair in the adult. Therefore, adult progenitors that mimic the "growth" differentiation profile during "repair" will lead to improved mechanical outcome. We have identified resident tendon progenitors in an inducible Cre model for alpha smooth muscle actin (aSMA-CreERT2) that contribute to scleraxis-expressing (ScxGFP) fibroblasts in the tendon midsubstance during growth and are also the main contributors to tendon healing from the paratenon in the adult. The SMACre model will be used in combination with four other GFP reporter mouse models that identify subpopulations within this lineage in the tendon midsubstance. We will define the expression profiles that delineate these subpopulations to map the heterogeneity of the tendon midsubstance. To better define the signaling pathways that regulate differentiation of these resident progenitors, we will begin by knocking out TGFß signaling in these cells to determine how this important pathway regulates normal cell turnover during growth and healing. Finally, using the expression profiles and markers defined in earlier studies, we will isolate analogous resident progenitors from human tendon tissue. The reparative potential of these human sources will be compared to their SMACre counterparts in a common test platform in the patellar tendon defect of immunodeficient NSG mice. If successful, this proposal will 1) help characterize the resident tendon progenitors that respond to injury, 2) help isolate progenitor cel sources conducive for tendon repair, and 3) provide biological success criteria for tenogenic differentiation. Finally, this research strategy under the guidance of my mentoring team will give me the experience needed to jump start a career in tendon biology and tissue engineering.

 描述（由申请人提供）：我的职业目标是成为一名独立的研究人员，有助于肌腱生物学的理解，并制定肌腱修复的改进策略。我的研究训练始于一名工程师，专注于开发使肌腱恢复正常机械功能的疗法。我意识到，对导致修复改善的细胞机制的不完整知识阻碍了我开发有效的治疗策略。我决定，我需要提高我对肌腱祖细胞起源的理解，定义其分化状态的标志物，以及在开发新的修复策略之前调节其分化的信号通路。我的导师团队由我的主要导师大卫罗博士和康涅狄格大学健康中心的其他熟练的肌肉骨骼生物学家和工程师组成，沿着还有一位外部肌腱发育生物学家，为我提供了一个特殊的环境来调查这些问题，并培养必要的技能，作为一名独立的调查员为该领域做出贡献。我们已经开发了一个剧目的转基因谱系追踪和GFP报告小鼠，确定居民肌腱祖细胞群体，并展示了异质性的肌腱midsubstance，曾经被认为是相当同质的细胞。本申请提出了一种策略，以表征驻留肌腱祖细胞的transciptomic和蛋白质组学谱，因为它们在肌腱生长和损伤后自然愈合的正常过程中分化。核心假设是，在生长过程中驱动肌腱祖细胞分化的分子机制对改善成人肌腱修复至关重要。因此，在“修复”期间模拟“生长”分化概况的成体祖细胞将导致改善的机械结果。我们已经在α平滑肌肌动蛋白（aSMA-CreERT 2）的诱导型Cre模型中鉴定了常驻肌腱祖细胞，其在生长期间有助于肌腱中间质中的巩膜轴表达（ScxGFP）成纤维细胞，并且也是成人腱旁肌腱愈合的主要贡献者。SMACre模型将与其他四种GFP报告基因小鼠模型组合使用，这些小鼠模型在肌腱中间物质中鉴定该谱系内的亚群。我们将定义描述这些亚群的表达谱，以绘制肌腱中间物质的异质性。为了更好地确定调节这些常驻祖细胞分化的信号传导途径，我们将开始通过敲除这些细胞中的TGF β信号传导来确定这一重要途径如何调节生长和愈合期间的正常细胞周转。最后，使用早期研究中定义的表达谱和标记，我们将从人肌腱组织中分离类似的常驻祖细胞。这些人源的修复潜力将在免疫缺陷NSG小鼠的髌腱缺损中的共同测试平台中与其SMACre对应物进行比较。如果成功，该提议将1）有助于表征对损伤有反应的驻留肌腱祖细胞，2）有助于分离有利于肌腱修复的祖细胞来源，以及3）提供肌腱分化的生物学成功标准。最后，在我的导师团队的指导下，这项研究策略将为我提供在肌腱生物学和组织工程领域开始职业生涯所需的经验。

项目成果

Human Subacromial Bursal Cells Display Superior Engraftment Versus Bone Marrow Stromal Cells in Murine Tendon Repair.

• DOI: 10.1177/0363546518802842
• 发表时间: 2018-12
• 期刊: The American journal of sports medicine
• 作者: Dyrna F;Zakko P;Pauzenberger L;McCarthy MB;Mazzocca AD;Dyment NA
• 通讯作者: Dyment NA