Identification of Periostin as a critical niche for myofibroblast dynamics and fibrosis during tendon healing.

Tendon injuries are a major clinical problem, with poor patient outcomes caused by abundant scar tissue deposition during healing. Myofibroblasts play a critical role in the initial restoration of structural integrity after injury. However, persistent myofibroblast activity drives the transition to fibrotic scar tissue formation. As such, disrupting myofibroblast persistence is a key therapeutic target. While myofibroblasts are typically defined by the presence of αSMA+ stress fibers, αSMA is expressed in other cell types including the vasculature. As such, modulation of myofibroblast dynamics via disruption of αSMA expression is not a translationally tenable approach. Recent work has demonstrated that Periostin-lineage (PostnLin) cells are a precursor for cardiac fibrosis-associated myofibroblasts. In contrast to this, here we show that PostnLin cells contribute to a transient αSMA+ myofibroblast population that is required for functional tendon healing, and that Periostin forms a supportive matrix niche that facilitates myofibroblast differentiation and persistence. Collectively, these data identify the Periostin matrix niche as a critical regulator of myofibroblast fate and persistence that could be targeted for therapeutic manipulation to facilitate regenerative tendon healing.

Identification of Periostin as a critical niche for myofibroblast dynamics and fibrosis during tendon healing.

Tendon injuries are a major clinical problem, with poor patient outcomes caused by abundant scar tissue deposition during healing. Myofibroblasts play a critical role in the initial restoration of structural integrity after injury. However, persistent myofibroblast activity drives the transition to fibrotic scar tissue formation. As such, disrupting myofibroblast persistence is a key therapeutic target. While myofibroblasts are typically defined by the presence of αSMA+ stress fibers, αSMA is expressed in other cell types including the vasculature. As such, modulation of myofibroblast dynamics via disruption of αSMA expression is not a translationally tenable approach. Recent work has demonstrated that Periostin-lineage (PostnLin) cells are a precursor for cardiac fibrosis-associated myofibroblasts. In contrast to this, here we show that PostnLin cells contribute to a transient αSMA+ myofibroblast population that is required for functional tendon healing, and that Periostin forms a supportive matrix niche that facilitates myofibroblast differentiation and persistence. Collectively, these data identify the Periostin matrix niche as a critical regulator of myofibroblast fate and persistence that could be targeted for therapeutic manipulation to facilitate regenerative tendon healing.

Increased Ca2+ signaling through CaV 1.2 induces tendon hypertrophy with increased collagen fibrillogenesis and biomechanical properties.

Tendons are tension-bearing tissues transmitting force from muscle to bone for body movement. This mechanical loading is essential for tendon development, homeostasis, and healing after injury. While Ca2+ signaling has been studied extensively for its roles in mechanotransduction, regulating muscle, bone, and cartilage development and homeostasis, knowledge about Ca2+ signaling and the source of Ca2+ signals in tendon fibroblast biology are largely unknown. Here, we investigated the function of Ca2+ signaling through CaV 1.2 voltage-gated Ca2+ channel in tendon formation. Using a reporter mouse, we found that CaV 1.2 is highly expressed in tendon during development and downregulated in adult homeostasis. To assess its function, we generated ScxCre;CaV 1.2TS mice that express a gain-of-function mutant CaV 1.2 in tendon. We found that mutant tendons were hypertrophic, with more tendon fibroblasts but decreased cell density. TEM analyses demonstrated increased collagen fibrillogenesis in the hypertrophic tendons. Biomechanical testing revealed that the hypertrophic tendons display higher peak load and stiffness, with no changes in peak stress and elastic modulus. Proteomic analysis showed no significant difference in the abundance of type I and III collagens, but mutant tendons had about two-fold increase in other ECM proteins such as tenascin C, tenomodulin, periostin, type XIV and type VIII collagens, around 11-fold increase in the growth factor myostatin, and significant elevation of matrix remodeling proteins including Mmp14, Mmp2, and cathepsin K. Taken together, these data highlight roles for increased Ca2+ signaling through CaV 1.2 on regulating expression of myostatin growth factor and ECM proteins for tendon collagen fibrillogenesis during tendon formation.

Increased Ca 2+ signaling through Ca V 1.2 induces tendon hypertrophy with increased collagen fibrillogenesis and biomechanical properties.

Tendons are tension-bearing tissues transmitting force from muscle to bone for body movement. This mechanical loading is essential for tendon development, homeostasis, and healing after injury. While Ca 2+ signaling has been studied extensively for its roles in mechanotransduction, regulating muscle, bone and cartilage development and homeostasis, knowledge about Ca 2+ signaling and the source of Ca 2+ signals in tendon fibroblast biology are largely unknown. Here, we investigated the function of Ca 2+ signaling through Ca V 1.2 voltage-gated Ca 2+ channel in tendon formation. Using a reporter mouse, we found that Ca V 1.2 is highly expressed in tendon during development and downregulated in adult homeostasis. To assess its function, we generated ScxCre;Ca V 1.2 TS mice that express a gain-of-function mutant Ca V 1.2 channel (Ca V 1.2 TS ) in tendon. We found that tendons in the mutant mice were approximately 2/3 larger and had more tendon fibroblasts, but the cell density of the mutant mice decreased by around 22%. TEM analyses demonstrated increased collagen fibrillogenesis in the hypertrophic tendon. Biomechanical testing revealed that the hypertrophic Achilles tendons display higher peak load and stiffness, with no changes in peak stress and elastic modulus. Proteomics analysis reveals no significant difference in the abundance of major extracellular matrix (ECM) type I and III collagens, but mutant mice had about 2-fold increase in other ECM proteins such as tenascin C, tenomodulin, periostin, type XIV and type VIII collagens, around 11-fold increase in the growth factor of TGF-ß family myostatin, and significant elevation of matrix remodeling proteins including Mmp14, Mmp2 and cathepsin K. Taken together, these data highlight roles for increased Ca 2+ signaling through Ca V 1.2 on regulating expression of myostatin growth factor and ECM proteins for tendon collagen fibrillogenesis during tendon formation.

Scleraxis-lineage cells are required for tendon homeostasis and their depletion induces an accelerated extracellular matrix aging phenotype.

Aged tendons have disrupted homeostasis, increased injury risk, and impaired healing capacity. Understanding mechanisms of homeostatic disruption is crucial for developing therapeutics to retain tendon health through the lifespan. Here, we developed a novel model of accelerated tendon extracellular matrix (ECM) aging via depletion of Scleraxis-lineage cells in young mice (Scx-DTR). Scx-DTR recapitulates many aspects of tendon aging including comparable declines in cellularity, alterations in ECM structure, organization, and composition. Single-cell RNA sequencing demonstrated a conserved decline in tenocytes associated with ECM biosynthesis in aged and Scx-DTR tendons, identifying the requirement for Scleraxis-lineage cells during homeostasis. However, the remaining cells in aged and Scx-DTR tendons demonstrate functional divergence. Aged tenocytes become pro-inflammatory and lose proteostasis. In contrast, tenocytes from Scx-DTR tendons demonstrate enhanced remodeling capacity. Collectively, this study defines Scx-DTR as a novel model of accelerated tendon ECM aging and identifies novel biological intervention points to maintain tendon function through the lifespan.

Impact of aging on tendon homeostasis, tendinopathy development, and impaired healing.

Aging is a complex and progressive process where the tissues of the body demonstrate a decreased ability to maintain homeostasis. During aging, there are substantial cellular and molecular changes, with a subsequent increase in susceptibility to pathological degeneration of normal tissue function. In tendon, aging results in well characterized alterations in extracellular matrix (ECM) structure and composition. In addition, the cellular environment of aged tendons is altered, including a marked decrease in cell density and metabolic activity, as well as an increase in cellular senescence. Collectively, these degenerative changes make aging a key risk factor for the development of tendinopathies and can increase the frequency of tendon injuries. However, inconsistencies in the extent of age-related degenerative impairments in tendons have been reported, likely due to differences in how "old" and "young" age-groups have been defined, differences between anatomically distinct tendons, and differences between animal models that have been utilized to study the impact of aging on tendon homeostasis. In this review, we address these issues by summarizing data by well-defined age categories (young adults, middle-aged, and aged) and from anatomically distinct tendon types. We then summarize in detail how aging affects tendon mechanics, structure, composition, and the cellular environment based on current data and underscore what is currently not known. Finally, we discuss gaps in the current understanding of tendon aging and propose key avenues for future research that can shed light on the specific mechanisms of tendon pathogenesis due to aging.

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

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.

Characterization of scar tissue biomechanics during adult murine flexor tendon healing.

Tendon injuries are very common and result in significant impairments in mobility and quality of life. During healing, tendons produce a scar at the injury site, characterized by abundant and disorganized extracellular matrix and by permanent deficits in mechanical integrity compared to healthy tendon. Although a significant amount of work has been done to understand the healing process of tendons and to develop potential therapeutics for tendon regeneration, there is still a significant gap in terms of assessing the direct effects of therapeutics on the functional and material quality specifically of the scar tissue, and thus, on the overall tendon healing process. In this study, we focused on characterizing the mechanical properties of only the scar tissue in flexor digitorum longus (FDL) tendons during the proliferative and early remodeling healing phases and comparing these properties with the mechanical properties of the composite healing tissue. Our method was sensitive enough to identify significant differences in structural and material properties between the scar and tendon-scar composite tissues. To account for possible inaccuracies due to the small aspect ratio of scar tissue, we also applied inverse finite element analysis (iFEA) to compute mechanical properties based on simulated tests with accurate specimen geometries and boundary conditions. We found that the scar tissue linear tangent moduli calculated from iFEA were not significantly different from those calculated experimentally at all healing timepoints, validating our experimental findings, and suggesting the assumptions in our experimental calculations were accurate. Taken together, this study first demonstrates that due to the presence of uninjured stubs, testing composite healing tendons without isolating the scar tissue overestimates the material properties of the scar itself. Second, our scar isolation method promises to enable more direct assessment of how different treatment regimens (e.g., cellular ablation, biomechanical and/or biochemical stimuli, tissue engineered scaffolds) affect scar tissue function and material quality in multiple different types of tendons.

Method development and characterization of chick embryo tendon mechanical properties.

Tendons are involved in multiple disorders and injuries, ranging from birth deformities to tendinopathies to acute ruptures. The ability to characterize embryonic tendon mechanical properties will enable elucidation of mechanisms responsible for functional tendon formation. In turn, an understanding of tendon development could inform approaches for adult and embryonic tendon tissue engineering and regenerative medicine. The chick embryo is a scientifically relevant model that we have been using to study Achilles (calcaneal) tendon development. Chick embryo calcaneal tendons are challenging to mechanically test due to small size and delicate nature, and difficulty distinguishing embryonic tendons from muscle and fibrocartilage using the naked eye. Here, we developed and implemented a "marking protocol" to identify and isolate calcaneal tendons at different stages of chick embryonic development. Mechanical testing of tendons isolated using the marking protocol revealed trends in mechanical property development that were not observed with tendons isolated by naked eye (eyeballing). Marked tendons exhibited non-linear increases in tensile modulus and ultimate tensile strength, whereas eyeballed tendons exhibited linear increases in the same properties, reflecting a need for the marking protocol. Furthermore, the tensile mechanical properties characterized for marked tendons are consistent with previously reported trends in cell length-scale mechanical properties measured using atomic force microscopy. This report establishes new methodology to enable tensile testing of chick embryo tendons and provides new information about embryonic tendon mechanical property development.

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.

Correction to: Cue-Signal-Response Analysis in 3D Chondrocyte Scaffolds with Anabolic Stimuli.

This erratum is to add the following paragraph in the Acknowledgement section.

Cue-Signal-Response Analysis in 3D Chondrocyte Scaffolds with Anabolic Stimuli.

Articular cartilage is an avascular connective tissue responsible for bearing loads. Cell signaling plays a central role in cartilage homeostasis and tissue engineering by directing chondrocytes to synthesize/degrade the extracellular matrix or promote inflammatory responses. The aim of this paper was to investigate anabolic, catabolic and inflammatory pathways of well-known and underreported anabolic stimuli in 3D chondrocyte cultures and connect them to diverse cartilage responses including matrix regeneration and cell communication. A cue-signal-response experiment was performed in chondrocytes embedded in alginate scaffolds subjected to a 9-day treatment with 7 anabolic cues. At the signaling level diverse pathways were measured whereas at the response level glycosaminoglycan (GAG) synthesis and cytokine releases were monitored. A significant increase of GAG was observed for each stimulus and well known anabolic phosphoproteins were activated. In addition, WNK1, an underreported protein of chondrocyte signaling, was uncovered. At the extracellular level, inflammatory and regulating cytokines were measured and DEFB1 and CXCL10 were identified as novel contributors to chondrocyte responses, both closely linked to TLR signaling and inflammation. Finally, two new pro-growth factors with an inflammatory potential, Cadherin-11 and MGP were observed. Interestingly, well-known anabolic stimuli yielded inflammatory responses which pinpoints to the pleiotropic roles of individual stimuli.