Formation of a functional tendon enthesis during development and healing

总结/摘要 肌腱损伤通常发生在其骨性附着点附近，需要手术修复肌腱与骨的连接。成果 然而，修复后的效果很差，并导致疼痛、再损伤和重复手术。肩袖修复，用于 例如，它是最常见的肩关节手术之一，但却有20-94%的失败率。的根源 这些糟糕的结果是缺乏附着点的再生，附着点是连接健康组织的专门组织。 肌腱和骨骼。与肌腱-骨愈合过程中形成的杂乱无章的疤痕相反，健康的 附着点在细胞表型、细胞外基质组成、矿物质含量和细胞周期中具有空间梯度。 力学性能这种功能分级允许两种材料之间有效地传递应力， 肌腱和骨骼，它们的机械性能有很大的不同。附着点是由细胞池形成的， 胎儿和出生后早期的发育与肌腱和软骨前体不同。我们之前 定义了这些Gli 1+附着点干细胞（ESCs）的谱系，并表明Gli 1+附着点干细胞的起始和矿化， 附着点需要hedgehog（Hh）信号传导。尽管这项工作，控制ESC的转录网络， 差异化仍然是难以捉摸的。更好地理解结束语所必需的发展线索 形成，特别是矿化，将有助于指导成人肌腱的新干细胞治疗方法- 骨修复目的1为研究终结干细胞分化的转录调控机制。Gli1- CreERT-mTmG小鼠将用于分离来自不同发育阶段的附着点细胞，并使用scRNAseq 将被用来定义他们的转录组。转录因子Klf 2/4和Runx 1将被检查为 ESC分化和矿化的假定调节剂。目标2将确定必要性和充分性 Gli 1+附着点干细胞用于附着点再生。愈合将使用我们的小鼠肩袖进行评估 肌腱止点损伤模型。Gli 1+附着点干细胞和Hh信号对于再生的必要性将 分别使用细胞消融和功能丧失模型进行测试。Gli+假体柄的充分性 细胞和Hh信号再生将通过将这些细胞递送到附着点损伤并使用 功能增益模型。这些研究将确定分子机制， 祖细胞形成并矿化附着点。结果将对未来的再生产生直接影响 肌腱-骨修复的策略

SUMMARY/ABSTRACT Tendon injuries often occur near their bony attachments, requiring surgical repair of tendon to bone. Outcomes after repair, however, are poor and result in pain, reinjury, and repeated surgeries. Rotator cuff repair, for example, is among the most common shoulder surgeries, yet is plagued by 20-94% failure rates. At the root of these poor outcomes is a lack of regeneration of the enthesis, the specialized tissue that connects healthy tendon and bone. In contrast to the disorganized scar that forms during tendon-to-bone healing, the healthy enthesis has spatial gradients in cell phenotypes, extracellular matrix composition, mineral content, and mechanical properties. This functional grading allows for effective transfer of stress between two materials, tendon and bone, with vastly different mechanical properties. The enthesis is formed by a pool of cells during fetal and early postnatal development that is unique from tendon and cartilage precursors. We previously defined the lineage of these Gli1+ enthesis stem cells (ESCs) and showed that initiation and mineralization of the enthesis requires hedgehog (Hh) signaling. Despite this work, the transcriptional network that controls ESC differentiation remains elusive. A better understanding of the developmental cues necessary for enthesis formation, and mineralization in particular, will help guide new stem cell treatment approaches for adult tendon- to-bone repair. Aim 1 will determine the transcriptional regulation of enthesis stem cell differentiation. Gli1- CreERT-mTmG mice will be used to isolate enthesis cells from different developmental stages and scRNAseq will be used to define their transcriptomes. The transcription factors Klf2/4 and Runx1 will be examined as putative regulators of ESC differentiation and mineralization. Aim 2 will determine the necessity and sufficiency of Gli1+ enthesis stem cells for enthesis regeneration. Healing will be evaluated using our mouse rotator cuff tendon enthesis injury model. The necessity of Gli1+ enthesis stem cells and Hh signaling for regeneration will be tested using cell ablation and loss-of-function models, respectively. The sufficiency of Gli+ enthesis stem cells and Hh signaling for regeneration will be tested by delivery of these cells to enthesis injuries and using gain-of-function models, respectively. These studies will identify the molecular mechanisms by which progenitor cells form and mineralize the enthesis. Results will have a direct impact on future regenerative strategies for tendon-to-bone repair.