Scleraxis-lineage cell depletion improves tendon healing and disrupts adult tendon homeostasis

Abstract

Despite the requirement for Scleraxis-lineage (ScxLin) cells during tendon development, the function of ScxLin cells during adult tendon repair, post-natal growth, and adult homeostasis have not been defined. Therefore, we inducibly depleted ScxLin cells (ScxLinDTR) prior to tendon injury and repair surgery and hypothesized that ScxLinDTR mice would exhibit functionally deficient healing compared to wild-type littermates. Surprisingly, depletion of ScxLin cells resulted in increased biomechanical properties without impairments in gliding function at 28 days post-repair, indicative of regeneration. RNA sequencing of day 28 post-repair tendons highlighted differences in matrix-related genes, cell motility, cytoskeletal organization, and metabolism. We also utilized ScxLinDTR mice to define the effects on post-natal tendon growth and adult tendon homeostasis and discovered that adult ScxLin cell depletion resulted in altered tendon collagen fibril diameter, density, and dispersion. Collectively, these findings enhance our fundamental understanding of tendon cell localization, function, and fate during healing, growth, and homeostasis.

Defining the activation profile and fate trajectory of adult Scleraxis-lineage cells during tendon healing by combining lineage tracing and spatial transcriptomics

“…The large presence of Scx GFP + continued in the bridging tissue at D21, though there was a progressive decrease in Scx GFP + and proximity to the tendon stubs increased (Fig 3E). These data are consistent with our previous hypothesis 5 that the contribution of adult Scx Ai9 cells are overwhelmed during early healing by an influx or expansion of a Scx+ population, followed by resolution of this population, and predominance of adult Scx Ai9 cells (particularly in the tendon stubs) during later healing.…”

“…Based on these data, and our previous work demonstrating that the broad Scx Lin cell pool (Scx-Cre) contributes to a persistent myofibroblast population 23 , we wanted to better define this population. Given that there are likely on-going ‘additions’ to this overall Scx Lin pool during healing 5 with cells turning on Scx expression at different times post-injury, we aimed to gain a more comprehensive understanding of the relationship between active Scx expression, adult Scx Ai9 cells and myofibroblast fate. To investigate this, we generated Scx Ai9 ; Scx GFP dual-reporter mice and traced these cells’ myofibroblast fate during tendon healing (Fig.…”

“…At 10-12 weeks of age, male and female mice underwent complete transection and repair of the flexor digitorum longus (FDL) tendon in the hind paw as previously described 5, 8, 9 . Mice were injected prior to surgery with 15-20μg of sustained-release buprenorphine and then anesthetized with Ketamine (100mg/kg) and Xylazine (10mg/kg).…”

“…Scleraxis ( Scx ), a basic helix-loop-helix transcription factor, is a critical regulator of tendon formation 1, 2 , with loss of Scx resulting in developmental deficits in tendon. In addition, Scx-lineage (Scx Lin ) cells play a critical role in tendon healing 3–5 , particularly in the formation of an organized cellular bridge between the injured tendon stubs 6 . However, defining the function of Scx Lin cells and their associated molecular programs are complicated by the heterogeneity of this cell lineage.…”

“…That is, the Scx Lin cells encompass those cells that expressed Scx at some point during embryonic development, the progeny of these cells, as well as any cell that expresses Scx during postnatal growth, adult homeostasis, and in response to injury. We have previously shown that depletion of Scx Lin cells in the adult flexor tendon improves the tendon healing and has only a modest impact on short-term tendon homeostasis 5 . Thus, we suggested that there are on-going ‘waves’ of cells being added to the Scx Lin populations throughout the healing process, with distinct Scx Lin subpopulations playing temporally- and spatially-dependent functions.…”