Defining the spatial-molecular map of fibrotic tendon healing and the drivers of Scleraxis-lineage cell fate and function.

Tendon injuries heal via a scar-mediated response, and there are no biological approaches to promote more regenerative healing. Mouse flexor tendons heal through the formation of spatially distinct tissue areas: a highly aligned tissue bridge between the native tendon stubs that is enriched for adult Scleraxis-lineage cells and a disorganized outer shell associated with peri-tendinous scar formation. However, the specific molecular programs that underpin these spatially distinct tissue profiles are poorly defined. In the present study, we combine lineage tracing of adult Scleraxis-lineage cells with spatial transcriptomic profiling to define the overarching molecular programs that govern tendon healing and cell-fate decisions. Pseudotime analysis identified three fibroblast trajectories (synthetic, fibrotic, and reactive) and key transcription factors regulating these fate-switching decisions, including the progression of adult Scleraxis-lineage cells through the reactive trajectory. Collectively, this resource defines the molecular mechanisms that coordinate the temporo-spatial healing phenotype, which can be leveraged to inform therapeutic candidate selection.

Metabolic Regulation of Tendon Inflammation and Healing Following Injury.

PURPOSE OF REVIEW: This review seeks to provide an overview of the role of inflammation and metabolism in tendon cell function, tendinopathy, and tendon healing. We have summarized the state of knowledge in both tendon and enthesis. RECENT FINDINGS: Recent advances in the field include a substantial improvement in our understanding of tendon cell biology, including the heterogeneity of the tenocyte environment during homeostasis, the diversity of the cellular milieu during in vivo tendon healing, and the effects of inflammation and altered metabolism on tendon cell function in vitro. In addition, the mechanisms by which altered systemic metabolism, such as diabetes, disrupts tendon homeostasis continue to be better understood. A central conclusion of this review is the critical need to better define fundamental cellular and signaling mechanisms of inflammation and metabolism during tendon homeostasis, tendinopathy, and tendon healing in order to identify therapies to enhance or maintain tendon function.

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

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.

Effects of tamoxifen on tendon homeostasis and healing: Considerations for the use of tamoxifen-inducible mouse models.

The use of tamoxifen-inducible models of Cre recombinase in the tendon field is rapidly expanding, resulting in an enhanced understanding of tendon homeostasis and healing. However, the effects of tamoxifen on the tendon are not well-defined, which is particularly problematic given that tamoxifen can have both profibrotic and antifibrotic effects in a tissue-specific manner. Therefore, in the present study, we examined the effects of tamoxifen on tendon homeostasis and healing in male and female C57Bl/6J mice. Tamoxifen-treated mice were compared to corn oil (vehicle)-treated mice. In the "washout" treatment regimen, mice were treated with tamoxifen or corn oil for 3 days beginning 1 week prior to undergoing complete transection and surgical repair of the flexor digitorum longus tendon. In the second regimen, mice were treated with tamoxifen or corn oil beginning on the day of surgery, daily through day 2 postsurgery, and every 48 hours thereafter (D0-2q48) until harvest. All repaired tendons and uninjured contralateral control tendons were harvested at day 14 postsurgery. Tamoxifen treatment had no effect on tendon healing in male mice, regardless of the treatment regimen, while Max load was significantly decreased in female repairs in the Tamoxifen washout group, relative to corn oil. In contrast, D0-2q48 corn oil treatment in female mice led to substantial disruptions in tendon homeostasis, relative to washout corn oil treatment. Collectively, these data clearly define the functional effects of tamoxifen and corn oil treatment in the tendon and inform future use of tamoxifen-inducible genetic models.

NF-κB activation persists into the remodeling phase of tendon healing and promotes myofibroblast survival.

Although inflammation is necessary during the early phases of tissue repair, persistent inflammation contributes to fibrosis. Acute tendon injuries often heal through a fibrotic mechanism, which impedes regeneration and functional recovery. Because inflammation mediated by nuclear factor κB (NF-κB) signaling is implicated in this process, we examined the spatial, temporal, and cell type-specific activation profile of canonical NF-κB signaling during tendon healing. NF-κB signaling was maintained through all phases of tendon healing in mice, including the remodeling phase, and tenocytes and myofibroblasts from the Scleraxis (Scx) lineage were the predominant populations that retained NF-κB activation into the late stages of repair. We confirmed persistent NF-κB activation in myofibroblasts in human tendon scar tissue. Deleting the canonical NF-κB kinase, IKKß, in Scx-lineage cells in mice increased apoptosis and the deposition of the matrix protein periostin during the late stages of tendon repair, suggesting that persistent NF-κB signaling may facilitate myofibroblast survival and fibrotic progression. Consistent with this, myofibroblasts in human tendon scar samples displayed enhanced prosurvival signaling compared to control tissue. Together, these data suggest that NF-κB may contribute to fibrotic tendon healing through both inflammation-dependent and inflammation-independent functions, such as NF-κB-mediated cell survival.

Deletion of NFKB1 enhances canonical NF-κB signaling and increases macrophage and myofibroblast content during tendon healing.

Flexor tendon injuries heal with excessive scar tissue that limits range of motion and increases incidence of re-rupture. The molecular mechanisms that govern tendon healing are not well defined. Both the canonical nuclear factor kappa B (NF-κB) and mitogen activated protein kinase (MAPK) pathways have been implicated in tendon healing. The gene NFKB1 (proteins p105/p50) is involved in both NF-κB and MAPK signaling cascades. In the present study, we tested the hypothesis that global NFKB1 deletion would increase activation of both NF-κB and MAPK through loss of signaling repressors, resulting in increased matrix deposition and altered biomechanical properties. As hypothesized, NFKB1 deletion increased activation of both NF-κB and MAPK signaling. While gliding function was not affected, NFKB1 deletion resulted in tendons that were significantly stiffer and trending towards increased strength by four weeks post-repair. NFKB1 deletion resulted in increased collagen deposition, increase macrophage recruitment, and increased presence of myofibroblasts. Furthermore, NFKB1 deletion increased expression of matrix-related genes (Col1a1, Col3a1), macrophage-associated genes (Adgre1, Ccl2), myofibroblast markers (Acta2), and general inflammation (Tnf). Taken together, these data suggest that increased activation of NF-κB and MAPK via NFKB1 deletion enhance macrophage and myofibroblast content at the repair, driving increased collagen deposition and biomechanical properties.

Cell non-autonomous functions of S100a4 drive fibrotic tendon healing.

Identification of pro-regenerative approaches to improve tendon healing is critically important as the fibrotic healing response impairs physical function. In the present study we tested the hypothesis that S100a4 haploinsufficiency or inhibition of S100a4 signaling improves tendon function following acute injury and surgical repair in a murine model. We demonstrate that S100a4 drives fibrotic tendon healing primarily through a cell non-autonomous process, with S100a4 haploinsufficiency promoting regenerative tendon healing. Moreover, inhibition of S100a4 signaling via antagonism of its putative receptor, RAGE, also decreases scar formation. Mechanistically, S100a4 haploinsufficiency decreases myofibroblast and macrophage content at the site of injury, with both cell populations being key drivers of fibrotic progression. Moreover, S100a4-lineage cells become α-SMA+ myofibroblasts, via loss of S100a4 expression. Using a combination of genetic mouse models, small molecule inhibitors and in vitro studies we have defined S100a4 as a novel, promising therapeutic candidate to improve tendon function after acute injury.

Scleraxis lineage cells contribute to organized bridging tissue during tendon healing and identify a subpopulation of resident tendon cells.

During tendon healing, it is postulated that tendon cells drive tissue regeneration, whereas extrinsic cells drive pathologic scar formation. Tendon cells are frequently described as a homogenous, fibroblast population that is positive for the marker Scleraxis (Scx). It is controversial whether tendon cells localize within the forming scar tissue during adult tendon healing. We have previously demonstrated that S100 calcium-binding protein A4 (S100a4) is a driver of tendon scar formation and marks a subset of tendon cells. The relationship between Scx and S100a4 has not been explored. In this study, we assessed the localization of Scx lineage cells (ScxLin) following adult murine flexor tendon repair and established the relationship between Scx and S100a4 throughout both homeostasis and healing. We showed that adult ScxLin localize within the scar tissue and organize into a cellular bridge during tendon healing. Additionally, we demonstrate that markers Scx and S100a4 label distinct populations in tendon during homeostasis and healing, with Scx found in the organized bridging tissue and S100a4 localized throughout the entire scar region. These studies define a heterogeneous tendon cell environment and demonstrate discrete contributions of subpopulations during healing. These data enhance our understanding and ability to target the cellular environment of the tendon.-Best, K. T., Loiselle, A. E. Scleraxis lineage cells contribute to organized bridging tissue during tendon healing and identify a subpopulation of resident tendon cells.

The cellular basis of fibrotic tendon healing: challenges and opportunities.

Tendon injuries are common and can dramatically impair patient mobility and productivity, resulting in a significant socioeconomic burden and reduced quality of life. Because the tendon healing process results in the formation of a fibrotic scar, injured tendons never regain the mechanical strength of the uninjured tendon, leading to frequent reinjury. Many tendons are also prone to the development of peritendinous adhesions and excess scar formation, which further reduce tendon function and lead to chronic complications. Despite this, there are currently no treatments that adequately improve the tendon healing process due in part to a lack of information regarding the contributions of various cell types to tendon healing and how their activity may be modulated for therapeutic value. In this review, we summarize recent efforts to identify and characterize the distinct cell populations involved at each stage of tendon healing. In addition, we examine the mechanisms through which different cell populations contribute to the fibrotic response to tendon injury, and how these responses can be affected by systemic factors and comorbidities. We then discuss gaps in our current understanding of tendon fibrosis and highlight how new technologies and research areas are shedding light on this clinically important and intractable challenge. A better understanding of the complex cellular environment during tendon healing is crucial to the development of new therapies to prevent fibrosis and promote tissue regeneration.

Deletion of EP4 in S100a4-lineage cells reduces scar tissue formation during early but not later stages of tendon healing.

Tendon injuries heal via scar tissue rather than regeneration. This healing response forms adhesions between the flexor tendons in the hand and surrounding tissues, resulting in impaired range of motion and hand function. Mechanistically, inflammation has been strongly linked to adhesion formation, and Prostaglandin E2 (PGE2) is associated with both adhesion formation and tendinopathy. In the present study we tested the hypothesis that deletion of the PGE2 receptor EP4 in S100a4-lineage cells would decrease adhesion formation. S100a4-Cre; EP4 flox/flox (EP4cKOS100a4) repairs healed with improved gliding function at day 14, followed by impaired gliding at day 28, relative to wild type. Interestingly, EP4cKOS100a4 resulted in only transient deletion of EP4, suggesting up-regulation of EP4 in an alternative cell population in these mice. Loss of EP4 in Scleraxis-lineage cells did not alter gliding function, suggesting that Scx-lineage cells are not the predominant EP4 expressing population. In contrast, a dramatic increase in α-SMA+, EP4+ double-positive cells were observed in EP4cKOS100a4 suggesting that EP4cKOS100a4 repairs heal with increased infiltration of EP4 expressing α-SMA myofibroblasts, identifying a potential mechanism of late up-regulation of EP4 and impaired gliding function in EP4cKOS100a4 tendon repairs.