Depletion of Scleraxis-lineage cells during tendon healing transiently impairs multi-scale restoration of tendon structure during early healing

Abstract

Tendons are composed of a heterogeneous cell environment, with Scleraxis-lineage (ScxLin) cells being the predominant population. Although ScxLin cells are required for maintenance of tendon homeostasis, their functions during tendon healing are unknown. To this end, we first characterized the spatiotemporal dynamics of ScxLin cells during tendon healing, and identified that the overall ScxLin pool continuously expands up to early remodeling healing phase. To better define the function of ScxLin cells during the late proliferative phase of healing, we inducibly depleted ScxLin cells from day 14–18 post-surgery using the Scx-Cre; Rosa-DTR mouse model, with local administration of diphtheria toxin inducing apoptosis of ScxLin cells in the healing tendon. At D28 post-surgery, ScxLin cell depleted tendons (DTRScxLin) had substantial impairments in structure and function, relative to WT, demonstrating the importance of ScxLin cells during tendon healing. Next, bulk RNAseq was utilized to identify the underlying mechanisms that were impaired with depletion and revealed that ScxLin depletion induced molecular and morphological stagnation of the healing process at D28. However, this stagnation was transient, such that by D56 tendon mechanics in DTRScxLin were not significantly different than wildtype repairs. Collectively, these data offer fundamental knowledge on the dynamics and roles of ScxLin cells during tendon healing.

Epitenon-derived cells comprise a distinct progenitor population that contributes to both tendon fibrosis and regeneration following acute injury

“…Given the spatially distinct localization of GLAST Ai9 epitenon-derived cells (peripheral capsule vs. organized bridging tissue), we next assessed the relationship between GLAST Ai9 cells and previously identified tenocyte populations (Scx-GFP+) 20, 23, 24 throughout healing. Prior to D10, GLAST Ai9 and Scx-GFP+ cells in and around the injury site remained largely distinct populations.…”

“…Mice then underwent complete transection and repair of the flexor digitorum longus (FDL) tendon in the hind paw as previously described. 22, 24, 50, 63–65 Briefly, mice were anesthetized with Ketamine (100mg/kg) and Xylazine (10mg/kg) and sustained-release buprenorphine (ZooPharm™; 0.5-1.0 mg/kg) was applied subcutaneously for post-operative pain management. To reduce chances of rupture at the repair site, the FDL tendon was first transected at the myotendinous junction (MTJ) and the skin was closed with non-resorbable 5-0 suture (#668G Ethilon, Ethicon Inc., Bridgewater, NJ, USA).…”

Preclinical tendon and ligament models: Beyond the 3Rs (replacement, reduction, and refinement) to 5W1H (why, who, what, where, when, how)

“…Previous studies on mouse flexor tendon investigated injuries in the plantar region of the paw at the metatarsals (zone III) where the tendons are larger, enabling the surgeon to suture the cut ends of the tendons together after laceration 113–118 . More recent experiments using this zone III model delineated novel molecular and cellular components of flexor tendon healing in a series of elegant experiments 119–123 . Although studies conducted outside of zone II have provided invaluable insights into the biology of flexor tendon scarring, the anatomy of the flexor tendons from the regions studied differs remarkably when compared with the flexor tendons of clinically‐relevant zone II.…”

“…[113][114][115][116][117][118] More recent experiments using this zone III model delineated novel molecular and cellular components of flexor tendon healing in a series of elegant experiments. [119][120][121][122][123] Although studies conducted outside of zone II have provided invaluable insights into the biology of flexor tendon scarring, the anatomy of the flexor tendons from the regions studied differs remarkably when compared with the flexor tendons of clinically-relevant zone II. However, a major limitation of the injury model in zone III is that the tendon is not encased in a synovial sheath, and therefore the cellular and molecular contributions of the sheath to peritendinous scarring adhesions are missing, which reduces the ability to simulate potentially important aspects of intrasynovial flexor tendon repair (Figure 8).…”

Skeletal Muscle Satellite Cells Co-Opt the Tenogenic GeneScleraxisto Instruct Regeneration

“…In adult tendon regeneration, the Tppp3 + Pdgfra + tendon stem cell population turn on Scx for tendon regeneration 10 . Lastly, Scx function is required in post-natal tendon growth and regeneration [11][12][13][14] .…”