Formation of a functional tendon enthesis during development and healing

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.