Matrix Biology

Muscle loading is required for embryonic tendon growth; however, the underlying mechanisms that regulate tendon development downstream of mechanical cues remain unidentified. Although tendons in muscle paralysis models are structurally and functionally inferior, whether these differences arise from cell or matrix deficits remains unclear. Analysis of muscular dysgenesis embryos by atomic force microscopy showed that structural and functional deficits in paralyzed tendon arise in part from reduced proliferation and collagen fibril disorganization. Bulk and single cell transcriptional analyses reveal that both collagenous and non-collagenous extracellular matrix components, as well as cytoskeletal and actomyosin-associated proteins, are dysregulated in mdg tendons, whereas tendon markers remain unchanged. Surprisingly, we find that an arrest of TGF{beta} signaling occurs during normal embryonic tendon growth and that TGF{beta} signaling is abnormally prolonged in paralyzed embryos. We also show for the first time, that specification of the epitenon depends on muscle contraction. Together, these findings establish cell and molecular requirements for muscle contraction in embryonic tendon development. TeaserMuscle contraction is required for embryonic tendon development through regulation of TGF{beta} signaling, epitenon formation, and matrix organization.

The innate immune system controls bacterial growth and modulates inflammation during wound healing. TCP-25 is a synthetic thrombin-derived host-defense peptide that combines direct antibacterial activity with neutralization of microbial products and modulation of CD14-dependent inflammatory signaling. We investigated whether this dual mechanism translates to human wounds using longitudinal samples from 24 healthy volunteers enrolled in a randomized, double-blind, within-participant, placebo-controlled phase I dose-escalation study of topical TCP-25 gel in matched epidermal suction blister wounds. We assessed inflammatory cytokines, neutrophil-derived proteins, wound exudation, cultivable bacterial burden, spatial bacterial distribution, and microbiome composition. TCP-25 reduced multiple cytokines, myeloperoxidase, and heparin-binding protein, with the strongest effects observed during the peak inflammatory phase. These changes were accompanied by reduced wound exudation and significant reductions in cultivable bacterial burden. Despite this antibacterial effect, microbiome composition and diversity remained largely unchanged, and participant-specific microbial profiles were preserved. TCP-25 therefore coordinated bacterial control, modulation of the physiological inflammatory response, and reduced wound leakage without major disruption of the resident microbiota composition. These findings provide clinical support for translating nature's endogenous host-defense principles into new therapies for complex wounds.

Elastin is the extracellular matrix (ECM) protein responsible for the elastic recoil property of certain tissues including the skin, arteries, and lung. Elastic fibre assembly begins with the coacervation of soluble monomeric tropoelastin and is driven by interactions with other ECM components such as glycosaminoglycans (GAGs). Previous research shows that GAG interactions can promote tropoelastin coacervation but lack structural and mechanistic details of this interaction. In this study, we describe the key interactions between tropoelastin and heparin using NMR spectroscopy and coacervation experiments. We propose a mechanism in which substoichiometric GAGs can act as a nucleating scaffold, primarily through transient multivalent interactions with domain 36 of tropoelastin, reducing the energetic barrier for coacervation. Our results provide the first detailed molecular view of tropoelastin-GAG interactions and support a role for negatively charged GAGs in modulating tropoelastin coacervation and thus initiating elastic fibre assembly.

Pressure injury induces progressive necrosis of skin and deep muscle, yet how mechanical loading reshapes molecular programs across tissues and time remains poorly defined. To address this gap, we established a rat pressure-injury model and performed longitudinal integrated profiling of skin and deep muscle across four stages, combining transcriptomics, proteomics and untargeted metabolomics with histological assessment. This multi-layered atlas revealed a staged injury program dominated by early transcriptional activation of innate immunity and complement-coagulation crosstalk, accompanied by neutrophil-associated responses and engagement of upstream regulatory networks. As injury advanced, a second axis of remodeling emerged, characterized by sustained suppression of mitochondrial energy metabolism and oxidative phosphorylation, particularly in muscle, together with extracellular matrix and adhesion rewiring. In parallel, skin preferentially activated barrier-repair and protein-homeostasis programs, including keratinization and autophagy-linked processes, indicating tissue-specific adaptation to the same mechanical insult. Temporal clustering and pathway-network analysis further showed that immune activation and metabolic collapse are not independent events but are dynamically coupled across stages, with muscle exhibiting broader molecular reprogramming and a stronger shift toward irreversible structural failure. Together, these data define a cross-tissue, stage-resolved molecular framework for pressure injury progression and identify pathway-level windows that may inform tissue-targeted and stage-specific intervention strategies.

The gut-joint axis describes how impaired intestinal epithelial function and increased gut permeability allow luminal factors to enter circulation. This can drive inflammation in Rheumatoid Arthritis, a chronic condition affecting the joint with systemic features. What mechanisms contribute to disease persistence are, as yet, incompletely understood. In health, extensively Oglycosylated intestinal mucins are central to epithelial protection and immune homeostasis; however, whether mucin glycosylation is altered during arthritis has not been addressed. Here, we investigated whether arthritisassociated inflammation alters mucin Oglycosylation, potentially compromising intestinal barrier function. Using a collageninduced arthritis mouse model, we combined epithelial transcriptomics, mass spectrometry-based glycomics, and imaging approaches to profile intestinal glycosylation. We identified distinct glycan remodeling in the colon, characterized by reduced fucosylation, while the ileum remained largely unaffected. In vitro studies using 3D human epithelial cultures further demonstrated that inflammatory cues, particularly from TNFactivated stromal cells, are sufficient to reduce epithelial fucosylation. Together, these findings identify a stromal-inflammatory mechanism that disrupts mucin glycosylation during arthritis. Loss of colonic fucosylation emerges as a novel element of inflammatory arthritis, providing an additional mechanistic link between intestinal inflammation and fibroblast-dependent modulation of the tissue microenvironment.

Tissue patterning is integral to development and regeneration, yet the factors that initiate morphogenetic patterning remain to be explored. Here, using embryonic chicken skin as a model, we show that perturbation of calcium signaling induces de novo feather bud formation in regions that normally do not form feather buds. This is achieved through coordinated changes in calcium dynamics, endogenous bioelectric currents, transcriptional regulation of calcium and potassium channel genes, and morphogen signaling. Different combinations of channel perturbations altered the number, distribution, size, and shape of induced feather buds. Live calcium imaging and extracellular electrophysiological recordings revealed homeostatic regulation, in which initially depressed calcium activity is followed by elevated calcium activity. Inward bioelectric currents emerge as de novo feather buds appear. Potassium channel blockade suppressed calcium dynamics, abolished endogenous currents, and inhibited new bud formation. Canonical feather morphogenesis pathways including Shh and {beta}-catenin are induced in these new buds. Our findings support a model in which developmental bioelectricity contributes to regulating the threshold of feather bud formation. These results identify developmental bioelectricity as an unrecognized regulatory layer of tissue patterning that warrants further study. Bullet points- Calcium signaling perturbation induces de novo feather bud formation in apteric skin - Ion channel perturbations regulate the formation, distribution and shape of new buds across a continuum, depending on channel type(s) and perturbation strength. - Elevated calcium activity and inward bioelectric currents accompany feather bud induction - Developmental bioelectricity represents an unrecognized regulatory layer for morphogenesis

BackgroundPhysical loading mediates postnatal growth, homeostasis, and healing of the tendon and its attachment to bone, which is critical for rotator cuff functional integrity. Our prior studies have highlighted the mechano-sensing role of primary cilia; However, the mechanisms through which cilia convert mechanical stimuli into structural functional adaptation under altered loading conditions remain unanswered. MethodsPublicly available scRNA-seq datasets of mechanically loaded human patellar tendon cells were re-analyzed to identify cilia-related transcriptional changes. Tendon-specific cilia knockout mice (ScxCre;Ift88fl/fl) and wild-type controls (Ift88fl/fl) underwent mechanical unloading induced by botulinum toxin A injection, followed by micro-computed tomography, biomechanical testing, histology, qPCR, and immunohistochemistry to evaluate structural, mechanical, and Hedgehog (Hh) signaling responses. Primary tendon fibroblasts from wild-type and cilia-deletion mice were treated with Hh agonist or antagonist to assess Hh signaling responsiveness in vitro. Students t-test for two groups and two-way ANOVA for two groups with two treatments were performed for our statistical analysis. ResultsHere, we find that mechanical force causes changes in cilia- and hedgehog (Hh)-related gene expression in human tendon fibroblasts. Cilia ablation in the enthesis blunts force-driven remodeling of tissue structure and mechanical strength. Cilia deletion also leads to impaired Hh signaling in tendon cells and decreased responsiveness to activation and inactivation of hedgehog signaling. ConclusionsOur results demonstrate loading-regulated ciliary Hh signaling during postnatal growth of the tendon and enthesis and provide proof-of-concept for developing new cilia-targeted mechanical and biological therapies for enthesis repair.

Systemic sclerosis (SSc) is an autoimmune connective tissue disease with pronounced sex differences: females are more frequently affected and males develop more severe skin fibrosis. The cellular mechanisms of this disparity remain unclear. Here we use single-cell transcriptomics of lesional, non-lesional, and healthy skin to define fibroblast states and sex-biased transcriptional programs during lesion development. We identify a sex-dependent divergence in SSc fibrotic regulation. Female fibroblasts exhibit heightened inflammatory signaling and canonical TGF-{beta}-driven extracellular matrix production, whereas male fibroblasts preferentially engage non-canonical TGF-{beta} pathways, mechanotransduction, and MYC-associated stress programs. We further reveal that the fibrotic lesional environment shows sex differences: SFRP2DPP4 fibroblasts predominate in females and COL11A1/COCH in males. Our findings uncover cellular mechanisms underlying sex differences in SSc fibrosis, highlight opportunities for sex-informed therapeutic strategies and underscore the necessity of integrating biological sex into precision medicine frameworks to identify divergent molecular drivers of fibrotic disease.

Fibroblasts mediate tissue repair after damage, but aberrant fibroblast behavior in response to injury can result in impaired wound healing. Severe burn injury often results in tissue scarring, but the underlying mechanisms by which fibroblasts respond to burn injury and the role of inflammation in fibrosis are not well understood. Here we developed fluorescent reporters of collagen expressing mesenchymal cells enabling real time imaging of fibroblasts during homeostatic development and in response to burn injury using larval zebrafish. We find that fibroblasts derived from the mesenchyme respond to burn injury by engaging in a maturation process, characterized by the expression of vimentin, which is reminiscent of larval development. In burned tissue, fibroblast maturation is perturbed by prolonged neutrophil infiltration, resulting in disorganized extracellular matrix (ECM) and delayed ECM remodeling, which can be rescued by neutrophil depletion. This work adds to our understanding of fibroblast development in zebrafish and shows that collagen expressing mesenchymal cells regulate ECM remodeling in coordination with immune cells during burn wound healing. Summary StatementVimentin-positive fibroblasts are required for normal wound healing in larval zebrafish, but the presence of inflammatory neutrophils after burn injury impairs fibroblast maturation and collagen remodeling.

The skin microbiome shapes local immunity, but the mechanisms of microbiome-immune crosstalk remain poorly understood. A major barrier to discovery is the lack of approaches that enable simultaneous, longitudinal measurement of microbes and immune cells from the same tissue without disrupting barrier integrity. Here we present a hydrogel-coated microneedle (MN) patch that enables minimally invasive co-sampling of viable microbes, immune cells, and interstitial fluid from skin. In humans, the patches were well tolerated and preserved inter-individual microbial signatures. Murine models colonized with commensal Staphylococcus epidermidis and the opportunistic pathogen Staphylococcus aureus, revealed distinct immune trajectories during commensal colonization, pathogen challenge, and commensal-pathogen co-colonization. Pathogen colonization drives progressive inflammatory amplification, whereas commensal exposure induces controlled immune activation that stabilizes over time. Notably, S. epidermidis reshapes pathogen-induced responses, producing a transient immune activation followed by attenuation of inflammation. These results establish MN sampling as a strategy to resolve immune-microbiome dynamics in barrier tissues and provide a framework for mechanistic studies of host-microbe interactions in health and disease.

An autocrine pro-adhesive/pro-contractile signaling loop, through the mechanosensitive transcriptional cofactor YAP, promotes fibrosis. The CCN family of matricellular proteins modify adhesive signaling. Of these, CCN3 is antifibrotic. We show that BLR-200, a CCN3-derived peptide, has anti-fibrotic properties in the bleomycin-induced model of scleroderma skin fibrosis. In vitro, BLR-200 delayed, but did not abolish, fibroblast adhesion to collagen and nuclear YAP localization. In vivo, BLR-200 prevented/treated bleomycin-induced skin fibrosis, and reduced bleomycin-induced expression of profibrotic genes including alpha-smooth muscle actin, CCN1 and CCN2. Lineage tracing and scRNA-seq analyses revealed that the myofibroblasts in this model were quantitatively derived from collagen-lineage Pi16+/Col15+ve fibroblasts. BLR-200 prevented myofibroblast differentiation in this model and trajectory of fibroblasts toward a Sfrp2-positive subset, a cell type associated with poor clinical outcome. BLR-200 impairs YAP activation in vitro and appearance of translationally-relevant fibroblast subtypes in vivo and is a novel anti-fibrotic agent for SSc skin fibrosis.

Glycine substitutions in the collagen triple helix cause diverse heritable disorders, but their effects vary with local sequence environment. We tested whether sequence context helps determine the consequences of Gly replacement by combining case-weighted bioinformatic analysis, thermodynamic measurements on collagen model peptides (CMPs), and all-atom molecular dynamics simulations. Case-weighted analysis of pathogenic COL3A1 glycine substitutions identified Pro immediately following the substituted Gly, corresponding to a GP context, as the strongest enriched local feature. To examine this experimentally, we designed CMPs with stabilizing terminal segments flanking native collagen sequence windows containing clinically observed Gly[->]Ser and Gly[->]Arg substitutions. Gly[->]Arg substitutions were generally more destabilizing than Gly[->]Ser at the same site. However, the strongest effect was sequence-dependent: within the Gly[->]Ser class, GP-site substitutions caused larger losses of thermal stability and unfolding enthalpy than nonGP substitutions, and some GP-site Gly[->]Ser mutations were as destabilizing as Gly[->]Arg substitutions. Molecular dynamics simulations showed that all peptides remained globally triple-helical, but GP-site mutants exhibited greater loss of canonical interchain hydrogen bonds and reduced local backbone accommodation. Thus, the effect of glycine substitution in collagen depends not only on the replacing residue but also on the permissiveness of the surrounding sequence, with Pro-adjacent sites representing especially restrictive local environments. Statement of SignificanceCollagen diseases often result from replacement of a required glycine in the triple helix, but the same substitution can have very different consequences at different sites. Using clinical bioinformatics, collagen model peptides, and molecular dynamics simulations, we show that the surrounding sequence is a major determinant of mutational outcome. The most restrictive context identified here is a GP site, in which proline immediately follows the substituted glycine: Gly[->]Ser mutations at GP sites are more destabilizing than Gly[->]Ser mutations at sites without a following proline and can be as damaging as Gly[->]Arg substitutions. These results define a sequence-based rule for collagen mutational tolerance relevant to both triple-helix stability and interpretation of pathogenic variants.

Mast cells (MCs) are tissue-resident sentinels of the innate immune system that play pivotal roles in host defense and inflammation. Perivascular MCs exert a particularly strong influence on the onset and dynamics of inflammation through the rapid, directional release of proinflammatory mediators into the circulation. Yet, the mechanisms governing their attachment to the vessel wall - a prerequisite for intravascular degranulation - remain poorly defined. Using a conditional knockout of integrin {beta}1 (Itgb1) in MCs, we investigated how perivascular positioning, degranulation, and vasoactive function contribute to inflammatory responses. In vivo imaging revealed that Itgb1 is essential for positioning MCs within the perivascular niche, particularly around arterioles. The absence of Itgb1 markedly reduced directional MC degranulation into blood vessels during skin inflammation. In vitro, Itgb1-deficient MCs displayed impaired degranulation kinetics together with altered SHIP1/PI3K-AKT signaling and calcium influx upon P2X7 ligation by ATP. During contact hypersensitivity, mice lacking Itgb1 in MCs exhibited strongly diminished ear swelling and reduced recruitment of multiple leukocyte subsets. Mechanistically, disordered MC positioning and attenuated degranulation impaired endothelial activation, resulting in decreased leukocyte adhesion and extravasation. These findings uncover a dual role for Itgb1 in regulating MC responsiveness and pro-inflammatory vasoactive function, establishing Itgb1-mediated perivascular MC positioning as a key prerequisite for effective leukocyte recruitment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/723399v1_ufig1.gif" ALT="Figure 1"> View larger version (80K): org.highwire.dtl.DTLVardef@ada618org.highwire.dtl.DTLVardef@73a85forg.highwire.dtl.DTLVardef@1330cccorg.highwire.dtl.DTLVardef@8d3b3c_HPS_FORMAT_FIGEXP M_FIG C_FIG

In AL amyloidosis, monoclonal immunoglobulin light chains (LCs) aggregate as amyloid fibrils in tissues. In synergy with the intrinsic aggregation propensity of specific LC sequences, microenvironment factors may be involved in tuning the disease pathophysiology, in particular proteolytic LC remodelling and heterotypic interactions in the extracellular milieu. Accounting for extrinsic modulators is critical for understanding the phenotypic variability of AL, usually imputed mainly to the LC diversity. We investigated the effects of apolipoprotein E (allele 3, apoE3) and clusterin (CLU), two amyloid-signature proteins involved in extracellular proteostasis, on the fibrillogenesis kinetics of amyloidogenic LC fragments from patient-derived sequences, as well as on aggregate composition, fibril morphology and thermodynamic stability. We show that apoE3 and CLU act as heterotypic interactors of prefibrillar and fibrillar LCs, significantly modulating LC amyloidogenesis, with complex and non-monotypic effects that range from anti- to pro-amyloidogenic depending on their concentration and on the LCs intrinsic amyloidogenicity. ApoE3 and CLU also influence fibril morphology, possibly by modifying protofilament association, and alter their thermodynamic properties. LC interactors may play a significant and insofar underappreciated role in the AL pathophysiology in vivo, likely contributing to phenotypic variability and structural polymorphisms and, possibly, to fibril resilience to amyloid reabsorption strategies.

In the context of muscle loss, bone repair is impaired, suggesting that muscle derived signals contribute to bone regeneration. However, how muscle surrounding the injury site communicates with the bone repair niche remains unclear. Here we found that CX3CL1 expression was induced in endothelial cells in muscle surrounding a femoral bone injury site. Deletion of Cx3cl1 impaired bone healing, demonstrating a functional role for CX3CL1 in bone repair. A CX3CL1 receptor, CX3CR1, was expressed by PDGFR stromal progenitors and lineage tracing showed that CX3CR1 expressing osteoprogenitor lineage cells accumulated at the injury site during repair. PDGFR stromal progenitors showed enhanced osteoblastogenesis in response to recombinant CX3CL1. In older mice, local CX3CL1 delivery increased PDGFRCX3CR1 osteoprogenitor accumulation and improved bone repair. These findings identify a muscle bone signaling pathway in which endothelial CX3CL1 promotes bone repair through CX3CR1 expressing osteoprogenitors.

Receptor activator of NF{kappa}B ligand (RANKL) is important for bone metabolism, but also modulates immune processes. We showed that mast cells (MCs) are involved in RANKL regulation, but the importance of MC-derived RANKL in skin inflammation has not yet been investigated. In contact hypersensitivity (CHS), the absence of MC-derived RANKL led to reduced skin inflammation due to impaired leukocyte infiltration and blood lymphopenia. Surprisingly, we observed a massive hyperplasia of the distant inguinal lymph nodes in the absence of MC-RANKL. Using adoptive transfers, flow cytometry and whole-mount 3D imaging, we demonstrated that this was not caused by structural maladaptation, but rather by the inability of lymphocytes to exit in a timely manner. Importantly, RANKL deletion in skin MCs only replicated the effect of LN hyperplasia and blood lymphopenia. Moreover, MCs were involved in serum sphingosine-1-phosphate (S1P) regulation during sensitization and challenge. Intravascular administration of S1P restored timely lymphocyte egress, demonstrating a MC-induced organ-spanning RANKL-S1P axis. Consequently, peripheral skin MC-derived RANKL is essential for the timely lymphocyte egress from distant LNs, which may have important implications for the targeted treatment of inflammatory skin diseases.

Fibroblasts are traditionally considered structural cells that maintain tissue homeostasis and facilitate repair. However, accumulating evidence suggests they also participate in innate immunity, although their pattern recognition capabilities remain incompletely characterized. Here, we systematically assessed the innate immune responses of commercially available primary human dermal fibroblasts from a male and a female donor. Fibroblasts were stimulated with a panel of microbe-associated molecular patterns (MAMPs) targeting various pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NODs), Alpha kinase 1 (ALPK1) and STING. Innate immune activation was quantified by measuring the nuclear translocation of NF-{kappa}B via high content microscopy and cytokines and chemokines secretion by ELISA; baseline PRRs expression was determined by quantitative PCR. Only a restricted subset of agonists, specifically E. coli LPS (TLR4), Poly I:C (TLR3 / RIG-I) and unexpectedly ADP heptose (ALPK1) induced robust NF-{kappa}B activation and secretion of the chemokines IL-8 and MCP-1. Apart from IL-6 and RANTES, which were produced exclusively following Poly I:C stimulation, pro-inflammatory cytokines (IL-1{beta}, TNF, IFN-{beta}) and the anti-inflammatory cytokine IL-10 remained undetectable. Consistent with this limited reactivity, qPCR of PRRs revealed basal expression of TLR4 and ALPK1, whereas most other receptors were expressed at very low or undetectable levels. Notably, NOD1 was highly expressed although no cell activation was observed with several NOD1 agonists. Dose-response analysis revealed surprisingly high sensitivity to LPS. In conclusion, primary human dermal fibroblasts exhibit a highly selective but sensitive innate immune response, largely restricted to chemokine production upon PRR activation. This unexpected dissociation between chemokine and cytokine responses suggests that fibroblasts function as sentinel cells in early skin defense, capable of detecting key microbial patterns at low concentrations, to orchestrate local immune surveillance. Further investigation into interindividual variability and context-dependent activation is needed.

The terminal differentiation of osteoblasts into osteocytes, the most abundant cell type in cortical bone, is critical for skeletal homeostasis. Osteocyte loss is a hallmark of bone aging and fragility, yet the mechanisms regulating osteocyte formation and survival are poorly understood. We show that inactivation of fibroblast growth factor receptor 1 (Fgfr1) in the mature osteoblast lineage results in extensive osteocyte death, identifying FGFR1 signaling as essential for osteocyte viability and bone integrity. Lineage tracing and analysis of endogenous and induced appositional bone formation revealed that newly embedded osteocytes fail to survive without FGFR1. These osteocytes exhibited ectopic expression of osteocalcin and podoplanin within sclerostin-positive, TUNEL-reactive lacunae, along with defective dendrite formation and disruption of the local lacunocanalicular network. RNA sequencing of cortical bone demonstrated reduced expression of extracellular matrix (ECM) genes and neuronal regulatory genes, while histological and ultrastructural analyses showed disorganized collagen fibrils, diminished osteoid, and abnormal mineralization. In vitro, FGF signaling in Ocy454 cells regulated gene programs involved in development, axon guidance, and bone ECM organization, highlighting a dual function for FGF signaling in which it controls both matrix-dependent and intrinsic cell differentiation mechanisms during the osteoblast-to-osteocyte transition. We propose that FGFR1 deficiency causes ECM disorganization and impaired dendrite formation, disrupting osteocyte communication with neighboring bone and vascular cells, ultimately leading to cell death. These findings establish FGFR signaling as a critical regulator of osteocyte differentiation, viability of bone-embedded osteocytes, and bone homeostasis. Summary StatementFGFR signaling has a profound effect on adult bone extracellular matrix that is vital to maintaining the viability and morphology of newly formed osteocytes, their lacunocanalicular network and the maintenance of bone homeostasis.

Adult mammals have limited capacity for tissue regeneration, where most injuries resolve through fibrotic scarring rather than functional tissue restoration1-4. Studies in regenerative vertebrate species, including amphibians, teleost fish, reptiles and mammals, have established that the innate immune system plays instructive roles in regeneration5-11, yet the role of adaptive immune cells and how the immune response distinguishes regenerative from non-regenerative injuries, remain poorly understood. The mouse digit tip provides a rare mammalian model of complete multi-tissue regeneration where distal amputation through the terminal phalanx (P3) triggers complete multi-tissue regrowth, whereas a more proximal amputation of the same bone results in fibrotic scarring12-16. Using an intravital multiphoton imaging approach capable of longitudinally tracking bone remodeling and immune cells in live mice17, we find that regulatory T cells (Tregs) are selectively recruited to regenerating but not scarring digit tips. Tregs localize first to sites of osteoclast-mediated bone resorption and persist at the bone surface when an expanding stromal progenitor pool, known as the blastema, initiates digit regrowth. Acute depletion of Tregs impairs bone resorption and subsequent bone regrowth. Mice lacking T and B cells or CD4+ and CD8+ T cells show similar bone remodeling defects, suggesting a dominant role for Tregs within the adaptive immune compartment in promoting mammalian digit tip regeneration. Treg depletion impairs regeneration through an IL-10-independent mechanism, pointing to a non-canonical effector program. Lastly, pharmacological blockade of the chemokine receptor CXCR4 reduces Treg recruitment to the bone compartment, diminishes bone-associated macrophage accumulation, and attenuates bone degradation in regenerative amputations. Together, these findings identify Tregs as essential regulators of bone remodeling during mammalian digit tip regeneration.

How cells and their organelles are positioned in three-dimensional, organ level, anatomical context, is rarely investigated. Here we focus on cells, centrioles and primary cilia in the growing limb. Through the ciliums mechanobiological role in skeletal development, we explored the mechanobiology of morphogenesis. A transgenic mouse model (Centrin 2-GFP.ARL13B-mCherry), combined with an image analysis pipeline, can map cellular size, positions and orientations, centriole position and ciliary axoneme orientation, all with respect to the anatomy of the epiphysis or growth plate. The line was crossed with an ift88fl/flCreERT2 line to enable ciliary ift88 deletion. We used limb immobilization, to test for a role of mechanical forces associated with ambulatory loading, in the organization of these elements and transcriptomics to understand the role of forces in regulating growth plate morphogenic programs. The pipeline can accurately quantify expected patterns of cell orientation and size through zones of the growth plate. Analysis across thousands of cells, through regions and zones of multiple murine growth plates, reveals cilia prevalence is increased in the periphery, highest in the resting zone in the outer limb, harboring stem cells. Cilia length is greatest in the hypertrophic cells about to die or transdifferentiate, as part of the formation of bone from cartilage by endochondral ossification. The inducible and cartilage-specific, deletion of ciliary gene ift88, alters cell orientation and sizes and reduces ciliation in the areas where endochondral ossification is most disrupted, the periphery and expanded hypertrophic zones, linking changes in structure to function. Most strikingly, centriole position, including that of the basal body, from which the ciliary axoneme is extended, is not preferentially organised. In contrast, cilia axoneme orientation is preferentially organised. Axonemes are directed posterior or anterior, 45 degrees to the axis of the limb, irrespective of their position, which is defined by basal body position. Immobilization of the limb for 2 weeks markedly alters the transcriptomic profile of the growth plate, with changes to size and orientation of cells and alterations in matrix and cytoskeletal profiles. Within altered genes, primary cilia genes themselves are regulated, including those indicative of altered cilia signaling such as hedgehog signaling. However, despite the role of cilia in mechanobiology of the growing limb, and ciliary signature within changes to loading of the limb, cilia orientation is unaltered by the removal of ambulatory associated forces. Patterns of ciliation in control and IFT88cKO mice help reconcile the previously observed anisotropic effects of cilia perturbation, focusing study on stem cell-resting chondrocytes and hypertrophy, when considering the mechanobiological role of cilia in limb development. Endochondral ossification is apparently highly sensitive to ambulatory loading at transcriptomic level, including effects on ciliary genes and signaling. A highly organized orientation of these putative antennae is governed by centriole position-independent mechanisms and is independent to changes to ambulatory loading, indicating a cell intrinsic mechanism. The resilient position of axonemes in the limb, points to mechano-regulatory mechanisms for how cilia integrate biophysical signals. We propose that predominant ventral or dorsal orientation at 45 degrees to horizonal plane but never parallel to cranial-chordal or medial-lateral axis, ensures multiple signal integration and avoids single signal blindness.