Quantification of Cartilage Poroelastic Material Properties Via Analysis of Loading-Induced Cell Death.

Articular cartilage (AC) is a load-bearing tissue that covers long bones in synovial joints. The biphasic/poroelastic mechanical properties of AC help it to protect joints by distributing loads, absorbing impact forces, and reducing friction. Unfortunately, alterations in these mechanical properties adversely impact cartilage function and precede joint degeneration in the form of osteoarthritis (OA). Thus, understanding what factors regulate the poroelastic mechanical properties of cartilage is of great scientific and clinical interest. Transgenic mouse models provide a valuable platform to delineate how specific genes contribute to cartilage mechanical properties. However, the poroelastic mechanical properties of murine articular cartilage are challenging to measure due to its small size (thickness ∼ 50 microns). In the current study, our objective was to test whether the poroelastic mechanical properties of murine articular cartilage can be determined based solely on time-dependent cell death measurements under constant loading conditions. We hypothesized that in murine articular cartilage subjected to constant, sub-impact loading from an incongruent surface, cell death area and tissue strain are closely correlated. We further hypothesized that the relationship between cell death area and tissue strain can be used-in combination with inverse finite element modeling-to compute poroelastic mechanical properties. To test these hypotheses, murine cartilage-on-bone explants from different anatomical locations were subjected to constant loading conditions by an incongruent surface in a custom device. Cell death area increased over time and scaled linearly with strain, which rose in magnitude over time due to poroelastic creep. Thus, we were able to infer tissue strain from cell death area measurements. Moreover, using tissue strain values inferred from cell death area measurements, we applied an inverse finite element modeling procedure to compute poroelastic material properties and acquired data consistent with previous studies. Collectively, our findings demonstrate in the key role poroelastic creep plays in mediating cell survival in mechanically loaded cartilage and verify that cell death area can be used as a surrogate measure of tissue strain that enables determination of murine cartilage mechanical properties.

Tracking subjects' strategies in behavioural choice experiments at trial resolution.

Investigating how, when, and what subjects learn during decision-making tasks requires tracking their choice strategies on a trial-by-trial basis. Here, we present a simple but effective probabilistic approach to tracking choice strategies at trial resolution using Bayesian evidence accumulation. We show this approach identifies both successful learning and the exploratory strategies used in decision tasks performed by humans, non-human primates, rats, and synthetic agents. Both when subjects learn and when rules change the exploratory strategies of win-stay and lose-shift, often considered complementary, are consistently used independently. Indeed, we find the use of lose-shift is strong evidence that subjects have latently learnt the salient features of a new rewarded rule. Our approach can be extended to any discrete choice strategy, and its low computational cost is ideally suited for real-time analysis and closed-loop control.

Mapping causal links between prefrontal cortical regions and intra-individual behavioral variability.

Intra-individual behavioral variability is significantly heightened by aging or neuropsychological disorders, however it is unknown which brain regions are causally linked to such variabilities. We examine response time (RT) variability in 21 macaque monkeys performing a rule-guided decision-making task. In monkeys with selective-bilateral lesions in the anterior cingulate cortex (ACC) or in the dorsolateral prefrontal cortex, cognitive flexibility is impaired, but the RT variability is significantly diminished. Bilateral lesions within the frontopolar cortex or within the mid-dorsolateral prefrontal cortex, has no significant effect on cognitive flexibility or RT variability. In monkeys with lesions in the posterior cingulate cortex, the RT variability significantly increases without any deficit in cognitive flexibility. The effect of lesions in the orbitofrontal cortex (OFC) is unique in that it leads to deficits in cognitive flexibility and a significant increase in RT variability. Our findings indicate remarkable dissociations in contribution of frontal cortical regions to behavioral variability. They suggest that the altered variability in OFC-lesioned monkeys is related to deficits in assessing and accumulating evidence to inform a rule-guided decision, whereas in ACC-lesioned monkeys it results from a non-adaptive decrease in decision threshold and consequently immature impulsive responses.

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.

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement.

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a localized fibrocartilage phenotype characterized by accumulation of glycosaminoglycan (GAG)-rich matrix and remodeling of the collagen network. While fibrocartilage is a normal feature in impinged regions of healthy tendons, excess GAG deposition and disorganization of the collagen network are hallmark features of tendinopathy. Accordingly, impingement is clinically recognized as an important extrinsic factor in the initiation and progression of tendinopathy. Nevertheless, the mechanobiology underlying tendon impingement remains understudied. Prior efforts to elucidate the cellular response to tendon impingement have applied uniaxial compression to cells and excised tendon explants in vitro. However, isolated cells lack a three-dimensional extracellular environment crucial to mechanoresponse, and both in vitro and excised explant studies fail to recapitulate the multiaxial strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region. Moreover, in vivo models of tendon impingement lack control over the mechanical strain environment. To overcome these limitations, we present a novel murine hind limb explant model suitable for studying the mechanobiology of Achilles tendon impingement. This model maintains the Achilles tendon in situ to preserve local anatomy and reproduces the multiaxial strain environment generated by impingement of the Achilles tendon insertion upon the calcaneus during passively applied ankle dorsiflexion while retaining cells within their native environment. We describe a tissue culture protocol integral to this model and present data establishing sustained explant viability over 7 days. The representative results demonstrate enhanced histological GAG staining and decreased collagen fiber alignment secondary to impingement, suggesting elevated fibrocartilage formation. This model can easily be adapted to investigate different mechanical loading regimens and allows for the manipulation of molecular pathways of interest to identify mechanisms mediating phenotypic change in the Achilles tendon in response to impingement.

Cycles of goal silencing and reactivation underlie complex problem-solving in primate frontal and parietal cortex.

While classic views proposed that working memory (WM) is mediated by sustained firing, recent evidence suggests a contribution of activity-silent states. Within WM, human neuroimaging studies suggest a switch between attentional foreground and background, with only the foregrounded item represented in active neural firing. To address this process at the cellular level, we recorded prefrontal (PFC) and posterior parietal (PPC) neurons in a complex problem-solving task, with monkeys searching for one or two target locations in a first cycle of trials, and retaining them for memory-guided revisits on subsequent cycles. When target locations were discovered, neither frontal nor parietal neurons showed sustained goal-location codes continuing into subsequent trials and cycles. Instead there were sequences of timely goal silencing and reactivation, and following reactivation, sustained states until behavioral response. With two target locations, goal representations in both regions showed evidence of transitions between foreground and background, but the PFC representation was more complete, extending beyond the current trial to include both past and future selections. In the absence of unbroken sustained codes, different neuronal states interact to support maintenance and retrieval of WM representations across successive trials.

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.

Frontal and temporal coding dynamics in successive steps of complex behavior.

Ventrolateral prefrontal cortex (vlPFC), dorsolateral prefrontal cortex (dlPFC), and temporal cortex (TE) all contribute to visual decision-making. Accumulating evidence suggests that vlPFC may play a central role in multiple cognitive operations, perhaps resembling domain-general regions of the human frontal lobe. We trained monkeys in a task calling for learning, retrieval, and spatial selection of rewarded target objects. Recordings of neural activity covered large areas of vlPFC, dlPFC, and TE. Results suggested a central role for vlPFC in each cognitive operation with strong coding of each task feature, while only location was strongly coded in dlPFC and current object identity in TE. During target selection, target location was communicated first from vlPFC to dlPFC, followed by extensive mutual support. In vlPFC, stimulus identities were independently coded in different task operations. The results suggest a central role for the inferior frontal convexity in controlling successive operations of a complex, multi-step task.

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.

Prefrontal and Medial Temporal Lobe Cortical Contributions to Visual Short-Term Memory.

A number of recent studies have indicated that the medial temporal lobe (MTL) plays a critical role in working memory (WM) and perception, but these results have been highly controversial given the traditional association of MTL with long-term memory. We review the research and highlight important factors that need to be considered in determining the role of MTL in WM including set-size of used stimuli and feature complexity and/or feature conjunctions/bindings embedded in those stimuli. These factors relate to hierarchical and, accordingly, domain-specific theories of functional organization within the temporal lobe. In addition, one must consider process-specific theories too, because two key processes commonly understood to contribute recognition memory, namely, recollection and familiarity, also have robust support from neurophysiological and neuroimaging research as to their functional dissociations within MTL. PFC has long been heavily implicated in WM; however, relatively less is known about how the PFC contributes to recollection and familiarity, although dynamic prefrontal coding models in WM may help to explain their neural mechanisms. The MTL and PFC are heavily interconnected and do not operate independently in underlying WM. We propose that investigation of the interactions between these two regions in WM, particularly their coordinated neural activities, and the modeling of such interactions, will be crucial for the advancing understanding of the neural mechanisms of WM.

Hydrogel Swelling-Mediated Strain Induces Cell Alignment at Dentin Interfaces.

Cell and tissue alignment is a defining feature of periodontal tissues. Therefore, the development of scaffolds that can guide alignment of periodontal ligament cells (PDLCs) relative to tooth root (dentin) surfaces is highly relevant for periodontal tissue engineering. To control PDLC alignment adjacent to the dentin surface, poly(ethylene glycol) (PEG)-based hydrogels were explored as a highly tunable matrix for encapsulating cells and directing their activity. Specifically, a composite system consisting of dentin blocks, PEG hydrogels, and PDLCs was created to control PDLC alignment through hydrogel swelling. PDLCs in composites with minimal hydrogel swelling showed random alignment adjacent to dentin blocks. In direct contrast, the presence of hydrogel swelling resulted in PDLC alignment perpendicular to the dentin surface, with the degree and extension of alignment increasing as a function of swelling. Replicating this phenomenon with different molds, block materials, and cells, together with predictive modeling, indicated that PDLC alignment was primarily a biomechanical response to swelling-mediated strain. Altogether, this study describes a novel method for inducing cell alignment adjacent to stiff surfaces through applied strain and provides a model for the study and engineering of periodontal and other aligned tissues.

Integrated neural dynamics for behavioural decisions and attentional competition in the prefrontal cortex.

In the behaving monkey, complex neural dynamics in the prefrontal cortex contribute to context-dependent decisions and attentional competition. We used demixed principal component analysis to track prefrontal activity dynamics in a cued target detection task. In this task, the animal combined identity of a visual object with a prior instruction cue to determine a target/nontarget decision. From population activity, we extracted principal components for each task feature and examined their time course and sensitivity to stimulus and task variations. For displays containing a single choice object in left or right hemifield, object identity, cue identity and decision were all encoded in population activity, with different dynamics and lateralisation. Object information peaked at 100-200 ms from display onset and was largely confined to the contralateral hemisphere. Cue information was weaker and present even prior to display onset. Integrating information from cue and object, decision information arose more slowly and was bilateral. Individual neurons contributed independently to coding of the three task features. The analysis was then extended to displays with a target in one hemifield and a competing distractor in the other. In this case, the data suggest that each hemisphere initially encoded the identity of the contralateral object. The distractor representation was then rapidly suppressed, with the final target decision again encoded bilaterally. The results show how information is coded along task-related dimensions while competition is resolved and suggest how information flows within and across frontal lobes to implement a learned behavioural decision.

Frontopolar cortex shapes brain network structure across prefrontal and posterior cingulate cortex.

Primate frontopolar cortex (FPC), occupied by area 10, sits atop a functional hierarchy of prefrontal cortical regions, yet little is known about its involvement in wider cortical networks. Here we examined resting-state-functional-connectivity (rsfc) in rhesus monkeys with intact or lesioned FPC to identify cortical regions associated with FPC. We present a network of FPC-specific regions of interest (ROIs), whose connectivity was affected by lesion of FPC but not by lesion of neighbouring prefrontal cortex (principal sulcus). This network comprised 'core ROIs' with direct anatomical connections to FPC, located in ventrolateral prefrontal cortex, posterior cingulate cortex, and superior temporal gyrus, and 'peripheral ROIs' well connected to the core network. We further show that the principle effect of a lesion to FPC was to cause a profound disturbance of the functional connectivity of posterior cingulate and ventrolateral prefrontal cortex. We therefore suggest that FPC, posterior cingulate and ventrolateral prefrontal cortex comprise a network of interacting cortical areas whose interactions may be critical for mediating the contribution of FPC to decision making.

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.

Altered TGFB1 regulated pathways promote accelerated tendon healing in the superhealer MRL/MpJ mouse.

To better understand the molecular mechanisms of tendon healing, we investigated the Murphy Roth's Large (MRL) mouse, which is considered a model of mammalian tissue regeneration. We show that compared to C57Bl/6J (C57) mice, injured MRL tendons have reduced fibrotic adhesions and cellular proliferation, with accelerated improvements in biomechanical properties. RNA-seq analysis revealed that differentially expressed genes in the C57 healing tendon at 7 days post injury were functionally linked to fibrosis, immune system signaling and extracellular matrix (ECM) organization, while the differentially expressed genes in the MRL injured tendon were dominated by cell cycle pathways. These gene expression changes were associated with increased α-SMA+ myofibroblast and F4/80+ macrophage activation and abundant BCL-2 expression in the C57 injured tendons. Transcriptional analysis of upstream regulators using Ingenuity Pathway Analysis showed positive enrichment of TGFB1 in both C57 and MRL healing tendons, but with different downstream transcriptional effects. MRL tendons exhibited of cell cycle regulatory genes, with negative enrichment of the cell senescence-related regulators, compared to the positively-enriched inflammatory and fibrotic (ECM organization) pathways in the C57 tendons. Serum cytokine analysis revealed decreased levels of circulating senescence-associated circulatory proteins in response to injury in the MRL mice compared to the C57 mice. These data collectively demonstrate altered TGFB1 regulated inflammatory, fibrosis, and cell cycle pathways in flexor tendon repair in MRL mice, and could give cues to improved tendon healing.