Joel D. Boerckel

Background: Pregnancy and lactation impose substantial demands on maternal calcium homeostasis, leading to pronounced skeletal remodeling during lactation followed by recovery after weaning. Although bone mass is largely returned at the tissue level after weaning, it remains unclear whether osteocyte-level remodeling exhibits a similarly reversible pattern. Osteocytes regulate mineral mobilization through perilacunar/canalicular remodeling (PLR), which is elevated during lactation. However, its spatial and temporal regulation in response to reproduction remains poorly defined. Objective/Hypothesis: This study aims to determine whether PLR-regulated lacunar remodeling during reproduction varies with osteocyte location and relative age. We hypothesized that osteocyte PLR-mediated lacunar change is spatially heterogeneous during lactation, varies with osteocyte location and relative age, and may persist after weaning. Methods: DXA and μCT were used to assess skeletal changes. Sequential fluorochrome labeling tracked mineral dynamics and defined osteocyte relative age. Osteocyte PLR activity was evaluated by MMP13 immunohistochemistry. Lacunar-canalicular structure (LCS) was assessed using Ploton silver nitrate staining, and spatially resolved lacunar morphology was quantified using high-resolution backscattered scanning electron microscopy (bSEM). Results: At the tissue level, reproduction induced distinct skeletal responses, characterized by reversible cortical bone loss and persistent trabecular deterioration. Cortical bone loss during lactation was spatially asymmetric and confined to the posterior cortex. Fluorochrome labeling further resolved surface-specific remodeling patterns during reproduction, including endocortical resorption at the posterior cortex and sustained deposition at anterior endocortical and posterior periosteal surfaces.At the cellular level, osteocyte PLR activity increased during lactation in WT mice and returned after weaning, whereas no changes were observed in cKO mice. Consistently, lacunar size increased during lactation and returned toward baseline after weaning in WT mice but remained unchanged in cKO mice.Spatially resolved analysis demonstrated that lacunar remodeling was heterogeneous across cortical thickness. At the anterior cortex, lacunar enlargement occurred near the endocortical surface during lactation and was reversible after weaning. In contrast, at the posterior cortex, lacunar enlargement occurred near the periosteal surface and persisted after weaning. These spatial patterns corresponded to cortical regions enriched with newly formed osteocytes, whereas pre-existing osteocytes exhibited minimal changes. This spatial heterogeneity was absent in cKO mice. Conclusion: Osteocyte PLR-mediated lacunar remodeling during reproduction is spatially heterogeneous and varies with osteocyte location and relative age. These findings demonstrate that recovery at the tissue level does not necessarily extend to the osteocyte microenvironment and identify osteocyte PLR-mediated lacunar remodeling as a spatially structured and potentially persistent component of reproductive skeletal adaptation. Together, these results highlight a previously unrecognized layer of maternal skeletal health.

Osteoporotic bone degeneration involves progressive deterioration of trabecular microarchitecture, yet most scaffold-based bone tissue engineering studies evaluate osteogenesis in structurally favorable architectures that poorly represent compromised bone environments. Here, we establish a degeneration-inspired Voronoi scaffold platform in which point spacing serves as a single tunable architectural parameter to model transitions from dense mechanically integrated to severely deteriorated trabecular-like microenvironments. Increasing point spacing from 1.25 to 2.5 mm progressively reduced scaffold connectivity and stiffness while shifting deformation behavior from distributed load transfer to localized stress concentration, as confirmed by finite element analysis and mechanical testing. Benchmarking against clinically reported HR-pQCT datasets from postmenopausal women demonstrated that the intermediate 1.75 mm point spacing scaffold represents a clinically relevant compromised trabecular-like state, whereas the 2.5 mm scaffold represents a more severely deteriorated architectural condition. These architecture-dependent mechanical and structural transitions directly regulated hMSC behavior, where high point spacing scaffolds reduced cytoskeletal organization, stress fiber density, and osteogenic mineralization, establishing an architecture-associated osteogenic dysfunction regime. Polydopamine (PDA) coating progressively enhanced cytoskeletal organization and mineralization within architecturally compromised scaffolds without altering scaffold geometry. To quantitatively assess biointerface-mediated functional recovery, a Mineralization Rescue Percentage (MRP) framework was introduced, demonstrating up to 43% restoration of architecture-associated mineralization loss following PDA coating. Collectively, this work establishes a clinically contextualized degeneration-to-rescue biomaterials framework that shifts current scaffold design paradigms beyond structurally favorable architectures toward systematic investigation and functional rescue of architecture-associated osteogenic dysfunction within compromised bone-like microenvironments. Statement of Significance: Most scaffold-based bone tissue engineering studies evaluate osteogenesis in structurally favorable architectures that poorly represent compromised bone microenvironments associated with osteoporosis. Here, a clinically contextualized Voronoi scaffold platform is established in which point spacing serves as a single tunable architectural parameter to model transitions from mechanically integrated to structurally deteriorated trabecular-like states. By decoupling architectural and surface biointerface effects, the study demonstrates that architectural deterioration alone can drive cytoskeletal disruption and osteogenic failure. Importantly, polydopamine-mediated surface engineering partially restored cytoskeletal organization and mineralization within architecturally compromised scaffolds without altering bulk geometry. A Mineralization Rescue Percentage (MRP) framework was further introduced to quantitatively assess biointerface-mediated functional recovery within degeneration-inspired scaffold microenvironments.

Engineering functional bone scaffolds can be enhanced by integrating biologically instructive nanoscale surface features (e.g., nanotopography and nanoroughness), micro-scale geometric cues (e.g., curvature and porosity), and macro-scale mechanical properties (e.g., bulk stiffness); however, these length scales are often optimized independently. Here, we present a multiscale design framework combining additive manufacturing of triply periodic minimal surface (TPMS) gyroid scaffolds with plasma-assisted nanoscale surface engineering to regulate osteogenesis. Controlled variation in strut thickness generates distinct architectural regimes with coupled changes in curvature, porosity, and compressive modulus, recapitulating key aspects of trabecular bone mechanics. Micro-computed tomography confirms trabecular bone-like features, while finite element modeling and compression testing reveal that thinner architectures (0.6 mm) exhibit curvature-preserving geometry and distributed stress profiles favorable for cellular interaction. A low-temperature plasma electroless reduction (PER) strategy enables controlled silver nanoparticle deposition, while polydopamine-mediated adhesion ensures uniform and cytocompatible coatings. Notably, PDA-AgNP-functionalized 0.6 mm scaffolds significantly outperform unmodified and AgNP-only groups, exhibiting enhanced cytoskeletal organization, stress fiber formation, matrix mineralization, and osteogenic gene expression. These findings demonstrate that coupling nanoscale biointerface features with micro- and macro-scale architecture produces a synergistic enhancement in osteogenesis, providing a design framework for functional bone scaffolds. Table of Content Graphics: A plasma-enabled strategy integrates 3D-printed scaffold architecture with nanoscale surface engineering to enhance bone formation. By combining tunable structural design with uniform nanoparticle coating, the study shows that optimal biological responses occur only when mechanical and surface cues act together, highlighting a synergistic multiscale approach for designing advanced biomaterials for bone regeneration.

The skin exhibits extraordinary plasticity, enabling it to adapt to mechanical changes in the environment. While transient deformations are accommodated without lasting structural effects, sustained mechanical stress induces durable tissue changes. To investigate if these responses are mediated by shifts in epidermal stem cell fate, we employed two-photon intravital imaging to visualize epidermal cells in live skin subjected to acute mechanical forces. Mechanical force triggered the formation of intracellular “stress” vesicles within epidermal stem cells that filled with extracellular fluid and progressively enlarged, deforming the nucleus. Lineage tracing analyses revealed that the extent of nuclear deformation can predict cell fate outcomes. Moreover, mechanical stress caused sustained elevation of intracellular calcium in basal epidermal stem cells, and conditional deletion of the mechanosensitive ion channel Piezo1 disrupted calcium dynamics and increased stress vesicle formation. Using human skin xenografts, we demonstrated that stress vesicles are conserved in mammalian skin. Together, these findings identify stress vesicles as key mediators linking mechanical stress, calcium signaling, and epidermal stem cell fate. Using two-photon intravital imaging, the authors show that mechanical stress in skin triggers fluid-filled “stress vesicles” in epidermal cells, altering Piezo1-dependent calcium signals to drive stem cell differentiation and protect tissue integrity.

Abstract The BMP/GDF branch of TGF-β signaling regulates diverse aspects of skeletal biology, from skeletal development to maintenance and repair. However, the complexity, redundancy, and pleiotropy of BMP/GDF signaling have hamstrung a genetic dissection of its activities in different cell types over time. Here, we tested the feasibility of a three-transgene system using CRISPR/Cas9 to conditionally mutate six target sites, two each in the receptor-mediated Smad1 , Smad5 , and Smad8 transcriptional effectors of BMP/GDF signaling. Briefly, we used Prx1- cre to activate a conditional Cas9 transgene by recombination in early limb bud mesenchyme; this endonuclease then complexes with gRNAs expressed from a polycistronic tRNA-gRNA array for targeted mutagenesis. Slower than expected accumulation of gRNA-directed mutations in each Smad produced an unexpected postnatal skeletal phenotype. Beginning around one month after birth, all animals developed hyperostosis on the surface of all long limb bones, which progressively worsened with age. This woven bone expansion occurred through proliferation of RUNX2+ osteoprogenitor cells in the cambium layer of the periosteum, producing an abundance of periosteal osteoblasts. Endosteal osteoblasts did not increase in number but increased their mineralizing activity. As a result, the marrow cavities narrowed, and the patella and carpal elements, which have no periosteum, increased internal bone mass without altering shape and size. Thus, while BMP/GDF signaling is known to promote early postnatal bone growth, these data support an additional homeostatic role during late postnatal osteogenesis by regulating both periosteal and endosteal osteoblasts. Although this genetically simple approach requires further optimization to improve efficiency, combining three transgenes produced more than 160 conditionally mutagenized animals with a fully penetrant and reproducible phenotype. This is an advance over traditional cre/lox systems that scale in complexity with the number of target loci, and it highlights the potential to model a wide range of genetically complex traits and disorders.

ABSTRACT Fetal movements exert mechanical forces that shape the developing skeleton. Conditions that impair fetal movement can cause skeletal defects, but interventions are limited. Here, we show that maternal wheel running exercise regulates fetal skeletal development in mice. In wild‐type fetuses, maternal exercise stimulated joint morphogenesis and bone development. These changes could not be fully explained by altered placental transport. Therefore, we next evaluated the effects of maternal exercise in the Splotch‐delayed (Sp d ) mouse model of fetal akinesia, which features intact maternofetal communication, but homozygous mutants lack contractile limb skeletal muscle. Maternal exercise substantially rescued fetal akinesia‐impaired joint and bone development and prevented disuse‐induced resorption of the deltoid tuberosity. Further, bioreactor mechanical stimulation of explanted Sp d limbs, which remove systemic factors, similarly stimulated joint morphogenesis. Together, these findings identify maternal exercise as a regulator of fetal skeletal development, provide a platform for studying skeletal developmental mechanobiology, and suggest potential therapeutic implications for maternal exercise in skeletal conditions caused by impaired fetal movement.

Cells in load-bearing tissues experience both solid deformation and interstitial fluid flow during physiological loading, but the mechanisms by which they integrate these poroelastic mechanical signals remain poorly understood. Here, we develop a porous, nanoarchitected 3D scaffold that allows simultaneous delivery and control of matrix strain and fluid shear stress. We validated the platform through cyclic loading experiments and simulations of fluid-structure interactions. In static, stress-free culture mechanical environments, osteoblast-like cells adopted shapes, cytoskeletal architectures, and focal adhesion patterns templated by the 3D scaffold geometry. Under cyclic compression, the combined influence of matrix deformation and induced fluid flow disrupted this alignment, producing disordered actin structures and reduced focal adhesion eccentricity. These changes emerged even under low-frequency loading, within the drained poroelastic regime, indicating a high sensitivity of cytoskeletal organization to fluid-solid coupling. Our findings establish a tractable and tunable platform to investigate how cells sense and respond to dynamic poroelastic mechanical environments in 3D.

Oxygen tension dynamically regulates stem cell fate and tissue regeneration, yet how local oxygen availability is controlled within the bone marrow niche remains poorly understood. While bone marrow injury, such as by bone fracture, disrupts marrow vasculature, the consequences for local oxygen tension remain unclear. Here, we show in mice that while the tissue oxygen tension in bone marrow is low (25 mmHg, ~4% O 2 ), intracellular oxygenation is heterogeneous, and erythroid cells are high in oxygen. Bone fracture elevates oxygen tension in the injured bone marrow (>55 mmHg, ~8%), which persists for over a week postinjury. This oxygen elevation results not from angiogenesis, but rather from localized expansion of erythroid precursor cells in the injured bone marrow. Injury-activated erythroid precursors synthesize hemoglobin and concentrate oxygen at the injury site; however, blocking transferrin receptor 1 (CD71)-mediated iron uptake impairs hemoglobin synthesis, reduces local oxygen levels, and enhances bone regeneration through increased angiogenesis and osteogenesis. Together, these findings identify erythroid precursors as active regulators of local oxygen availability in the bone marrow niche, which may be targetable to enhance tissue regeneration.

Vascular morphogenesis requires persistent endothelial cell motility that is responsive to diverse and dynamic mechanical stimuli. Here, we interrogated the mechanotransductive feedback dynamics that govern endothelial cell motility and vascular morphogenesis. We show that the transcriptional regulators, YAP and TAZ, are activated by mechanical cues to transcriptionally limit cytoskeletal and focal adhesion maturation, forming a conserved mechanotransductive feedback loop that mediates human endothelial cell motility in vitro and zebrafish intersegmental vessel (ISV) morphogenesis in vivo. This feedback loop closes in 4 hours, achieving cytoskeletal equilibrium in 8 hours. Feedback loop inhibition arrested endothelial cell migration in vitro and ISV morphogenesis in vivo. Inhibitor washout at 3 hrs, prior to feedback loop closure, restored vessel growth, but washout at 8 hours, longer than the feedback timescale, did not, establishing lower and upper bounds for feedback kinetics in vivo. Mechanistically, YAP and TAZ induced transcriptional suppression of RhoA signaling to maintain dynamic cytoskeletal equilibria. Together, these data establish the mechanoresponsive dynamics of a transcriptional feedback loop necessary for persistent endothelial cell migration and vascular morphogenesis.

Vascular morphogenesis requires persistent endothelial cell motility that is responsive to diverse and dynamic mechanical stimuli. Here, we interrogated the mechanotransductive feedback dynamics that govern endothelial cell motility and vascular morphogenesis. We show that the transcriptional regulators, YAP and TAZ, are activated by mechanical cues to transcriptionally limit cytoskeletal and focal adhesion maturation, forming a conserved mechanotransductive feedback loop that mediates human endothelial cell motility in vitro and zebrafish intersegmental vessel (ISV) morphogenesis in vivo. This feedback loop closes in 4 hours, achieving cytoskeletal equilibrium in 8 hours. Feedback loop inhibition arrested endothelial cell migration in vitro and ISV morphogenesis in vivo. Inhibitor washout at 3 hrs, prior to feedback loop closure, restored vessel growth, but washout at 8 hours, longer than the feedback timescale, did not, establishing lower and upper bounds for feedback kinetics in vivo. Mechanistically, YAP and TAZ induced transcriptional suppression of RhoA signaling to maintain dynamic cytoskeletal equilibria. Together, these data establish the mechanoresponsive dynamics of a transcriptional feedback loop necessary for persistent endothelial cell migration and vascular morphogenesis.

The design of tunable hard and soft lattice scaffolds is key to advancing multiscale tissue engineering. In this study, we computationally designed and 3D-printed gyroid and diamond polylactic acid (PLA) scaffolds with varying lattice thicknesses and infills to modulate mechanical properties. Compression testing revealed a linear increase in modulus with increasing gyroid thickness (82–405 MPa), while diamond lattices with simple and body-centered infills reached up to 150 MPa, enabling tuning for both low- and high-density trabecular bone. Micro-CT analysis confirmed architectural fidelity, with scaffold porosity ranging from 63 to 85%, trabecular spacing (Tb.Sp) between 1.5 and 2.4 mm, and bone surface-to-volume ratios (BS/BV) of 3.2–6.4 mm 2 /mm 3, suggesting tunability toward native trabecular bone. Surface modification with polydopamine (PDA) enhanced scaffold bioactivity, supporting robust human bone marrow-derived mesenchymal stem cell (hMSC) attachment, spreading, and stress fiber formation. Importantly, preliminary osteogenic evaluation revealed enhanced mineral deposition in PDA-coated scaffolds compared to uncoated PLA, with PDA-coated diamond architectures exhibiting the highest calcium deposition relative to both gyroid and uncoated diamond scaffolds. These results demonstrate that osteogenic potential can be tuned through both topology and surface modification. In parallel, soft scaffolds were developed by reinforcing alginate hydrogels with hydroxyapatite (HAP) nanocrystals and 3D bioprinting them into gyroid, hexagonal, and square honeycomb geometries. Rheological testing confirmed improved shear-thinning and print fidelity with increasing HAP content. Cell encapsulation studies with fibroblasts revealed scaffold-dependent differences, where Alamar Blue and PicoGreen assays demonstrated the highest metabolic activity and DNA content in the square honeycomb design, followed by hexagonal and gyroid lattices. Together, these findings establish a framework in which lattice geometry, material reinforcement, and surface biofunctionalization can be systematically combined to create tunable scaffolds for both load-bearing and soft tissue applications, laying the groundwork for hybrid systems with spatial and mechanical gradients to regenerate complex tissues.

During development, fetal movement provides mechanical forces necessary for proper skeletal morphogenesis. Clinical conditions that limit fetal movement (i.e., fetal akinesia) can impair bone development and cause skeletal deformities. Based on this, we hypothesized that extra-embryonic mechanical stimulation could enhance fetal skeletal development. To mechanically stimulate embryos in utero, we performed maternal wheel running exercise. Briefly, female C57BL/6 mice were individually housed and acclimated to home cage running wheels for at least 2 weeks. Acclimated female mice were mated with male C57BL/6 mice, then housed without wheels until embryonic day (E) 13.5. At E13.5, pregnant mice were either re-housed with wheels from E13.5 to E16.5, inclusive (Ad Libitum), or subjected to 1 hour of daily supervised wheel running during the same period (Supervised). Control groups with locked wheels were included for both conditions (Jammed). Pregnant mice ran more consistently using supervised running therefore, we used this approach for future analyses. Embryos were harvested from Supervised and Jammed groups at E17.5 to quantify bone morphogenesis and placental transport efficiency. Forelimb bone morphogenesis was quantified using microcomputed tomography (voxel size = 3 µm, x-ray tube potential = 70 kVp, x-ray intensity = 145 µA, integration time = 300 ms). Embryos from exercised mothers showed increased humerus bone collar length, indicating that maternal exercise has osteogenic effects on developing embryos (Fig 1B). Intrauterine growth is also influenced by placental transport efficiency. To assess whether maternal exercise influences placental transport, we quantified the standard measurement of fetus weight to placental weight ratio (FW:PW). We observed no significant differences in FW:PW in response to Supervised maternal exercise. Together, our results demonstrate that maternal wheel running exercise promotes bone formation in fetal skeletons, independent of placental transport. Future studies will elucidate the underlying cellular and molecular mechanisms. Ultimately, this research may indicate maternal exercise as a non-invasive, in utero intervention for skeletal deformities caused by limited fetal movement. Additionally, this work establishes maternal exercise as a model for studying developmental mechanobiology in vivo. For any figures or tables, please contact the authors directly.

Long bone fractures not only disrupt the bone matrix but also injure the bone marrow. Fractures compromise the vasculature, and since bone marrow is a low-oxygen tissue, it has long been thought that disruption of arterial oxygen transport creates a severely hypoxic environment at the fracture site. However, in a previous study, we discovered that the early fracture gap is not hypoxic but instead exhibits high oxygen tension. In this study, we aimed to elucidate the underlying mechanism and leverage this new understanding to develop novel therapeutic approaches. Red blood cells (RBCs), which transport oxygen through the bloodstream, are produced in the bone marrow through erythropoiesis. RBC development begins with megakaryocytic-erythroid progenitors (MEPs), which undergo several stages of erythroid maturation, including the proerythroblast (ProE) and erythroblast (EryB) stages, before entering the circulation as enucleated reticulocytes. We performed femoral fracture (osteotomy) surgeries on adult female C57BL/6J mice using external fixation. All procedures were conducted in accordance with IACUC regulations (protocol no: 806482). Mice were euthanized three days post-fracture, and tissue samples were either embedded or cells were isolated by flushing the fracture site (between the middle pins). Intact contralateral bone marrow was used as a comparison. Tissues and cells were analyzed using multiplex histology, flow cytometry, and scRNAseq. Our findings revealed an increased abundance of CD71+ ProE at the fracture site. These cells are capable of locally regulating oxygen levels through the transcriptional regulation of mitochondrial heme synthesis for hemoglobin production. CD71 (transferrin receptor 1) is a marker for erythrocyte precursors and is essential for iron import into erythroid cells. CD71 blockade significantly reduced local oxygen tension while enhancing osteoprogenitor activation, angiogenesis, and bone formation in the callus and fracture gap. In this study, we demonstrate that erythroid progenitors are locally activated in the injured bone marrow and play a critical role in concentrating oxygen at the fracture site, which can be targeted to promote bone repair. Our findings establish that bone fractures also disrupt bone marrow homeostasis activating local erythropoiesis, providing new opportunities for future research and therapeutic strategies.

Vascularization via sprouting angiogenesis is crucial in bone development and fracture repair, occurring through endochondral ossification. Newly formed vessels invade the initially formed cartilage template to promote mineralization and transition to bone. Despite its importance, the precise mechanisms underlying vascular invasion remain elusive. Hypertrophic chondrocytes, which produce vascular endothelial growth factor (VEGF), are implicated in driving vascularization. Conversely, suppression of vascular invasion has been linked to enhanced chondrogenesis and delayed bone formation and healing. To elucidate these mechanisms, we developed an in vitro three-dimensional co-culture system combining GFP-labeled human umbilical vein endothelial cells (HUVECs) and human bone marrow stromal cells (hMSCs). Here we exemplify the integration of this model in two basic research projects focusing on (i) the functional significance of the chemokine CXCL12, also known as stromal-derived factor 1 (SDF-1), in YAP/TAZ-mediated angiogenesis and (ii) the effect of the matricellular growth factor Cyr61 during vascularized fracture repair. In short, depletion of YAP/TAZ from hMSCs impaired tubular network formation, while supplementation with recombinant CXCL12 rescued this phenotype, providing crucial evidence for a functional link during bone development. In addition, Cyr61 treatment significantly enhanced tubular network formation, suggesting its role in vascular morphogenesis and a potential strategy for future therapeutic approaches. Thus, our in vitro model provides insights into the complex interplay between endothelial cells and stromal cells during vascular invasion in bone development and repair. Understanding these mechanisms could inform novel therapeutic strategies for enhancing bone regeneration and fracture healing.

Cell-cell fusion is an evolutionarily conserved process that is essential for many functions, including the formation of bone-resorbing multinucleated osteoclasts. Osteoclast multinucleation involves dynamic interactions between the actin cytoskeleton and the plasma membrane that are still poorly characterized. We found that moesin, a cytoskeletal linker protein member of the Ezrin, radixin, and moesin (ERM) protein family, plays a critical role in both osteoclast fusion and function. Moesin inhibition favors osteoclast multinucleation as well as HIV-1- and inflammation-induced cell fusion. Accordingly, moesin depletion decreases membrane-to-cortex attachment and enhances the formation of tunneling nanotubes, F-actin-based intercellular bridges triggering cell-cell fusion. In addition, moesin regulates the formation of the sealing zone, a key structure determining osteoclast bone resorption area, and thus controls bone degradation via a β3-integrin/RhoA/SLK pathway. Finally, moesin-deficient mice have reduced bone density and increased osteoclast abundance and activity. These findings provide a better understanding of cell-cell fusion and osteoclast biology, opening new opportunities to specifically target osteoclasts in bone disease therapy.

Bone fracture repair initiates by periosteal expansion. The periosteum is a bilayered tissue composed of inner cambium and outer fibrous layers. Typically quiescent, periosteal progenitor cells proliferate upon fracture; however, the underlying transcriptional mechanisms remain unclear. Here, we show that deletion of the transcriptional regulators, yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ), from Osterix-expressing cells, which reside in the cambium, impairs periosteal expansion. YAP activation increases chromatin accessibility, preferentially at TEA domain transcription factor (TEAD) binding sites, and regulates both cell-intrinsic and cell-extrinsic cellular functions. We identify bone morphogenetic protein 4 ( Bmp4 ) as a YAP-TEAD target gene expressed in the cambium. In YAP/TAZ knockout mice, BMP4 delivery increased periosteal expansion through matrix accumulation and fibrous layer cell proliferation. Conversely, in wild-type mice, BMP4 delivery increased osteogenic activity and angiogenesis. Together, these data identify YAP-mediated transcriptional programs that promote layer-specific periosteal expansion.

Abstract Cells in load-bearing tissues experience both solid deformation and interstitial fluid flow during physiological loading, but the mechanisms by which they integrate these biphasic mechanical signals remain poorly understood. Here, we develop a porous, nanoarchitected 3D scaffold that allows simultaneous delivery and control of matrix strain and fluid shear stress. We validated the platform through fatigue loading experiments and simulations of fluid–structure interactions. In static culture, osteoblast-like cells adopted shapes, cytoskeletal architectures, and focal adhesion patterns templated by scaffold geometry. Under cyclic compression, the combined influence of matrix deformation and induced fluid flow disrupted this alignment, producing disordered actin structures and reduced focal adhesion eccentricity. These changes emerged even under low-frequency loading, within the drained poroelastic regime, indicating a high sensitivity of cytoskeletal organization to fluid-solid coupling. Our findings establish a tractable and tunable platform to investigate how cells sense and respond to dynamic biphasic mechanical environments in 3D. Significance Statement Cells in tissues such as bone experience mechanical inputs from both matrix deformation and interstitial fluid flow. However, existing in vitro systems often isolate one type of input or lack the ability to control both independently. We engineered a nanoarchitected 3D scaffold that delivers tunable biphasic mechanical inputs by combining structural compression and fluid flow. Without external loads, cells align their cytoskeleton and focal adhesions to the scaffold geometry. When subjected to dynamic loading, they transition to disordered morphologies and less mature focal adhesions, suggesting a transition to migratory states. These results highlight the sensitivity of cells to even subtle biphasic cues and provide a new platform to study how cells integrate multiple mechanical signals in 3D environments.

Clinically, compromised fracture healing often occurs at sites with less muscle coverage and muscle flaps can provide the necessary healing environment for appropriate healing in severe bone loss. However, the underlying mechanisms are largely unknown. Here, we established a mouse reporter model for studying muscle cell contribution to bone fracture repair. Analyzing skeletal muscle scRNA-seq datasets revealed that Prg4 marks a fibroadipogenic progenitor (FAP) subpopulation. In mice, Prg4 + cells were specifically located in the skeletal muscle, but not at the periosteum or inside cortical bone. These cells expressed FAP markers, responded to muscle injury, and became periosteal cells under normal and muscle injury conditions. Fracture fragmented muscle fibers, rapidly expanded Prg4 + FAPs at the injury site and promoted their migration into the fracture gap. Later, they gave rise to many chondrocytes, osteoblasts, and osteocytes in the outer periphery of callus next to muscle. In repaired bones, the descendants of Prg4 + FAPs were detected as mesenchymal progenitors in the periosteum and osteocytes at the prior fracture site. A second fracture activated those cells and stimulated them to become osteoblasts in the inner part of callus. Importantly, ablation of Prg4 + FAPs impaired fracture healing and functional repair. In an intramembranous bone injury model (drill-hole), Prg4 + FAPs became periosteal cells, but their contribution to bone defect repair was significantly less than in fractures. Taken together, we demonstrate the critical role of FAPs in endochondral bone repair and uncover a mechanism by which mesenchymal progenitors transform from muscle to cortical bone.

Abstract Fetal movements exert mechanical forces that shape the developing skeleton. Conditions that impair fetal movements can cause skeletal defects, but interventions are limited. Here, we show that maternal wheel running exercise regulates fetal skeletal development in mice. In wild-type fetuses, maternal exercise stimulated joint and bone morphogenesis. We reasoned that these effects occurred through either indirect maternofetal communication or direct mechanical stimulation of the fetus. Maternal exercise did not alter placental measures of nutrient transport. However, in the Splotch-delayed (Sp d ) mouse model of fetal akinesia, which features intact maternofetal communication but lacks fetal movements, maternal exercise substantially rescued fetal akinesia-impaired joint and bone development and prevented disuse-induced resorption of the deltoid tuberosity. Further, direct mechanical stimulation of Sp d limbs explanted from systemic factors similarly stimulated joint morphogenesis. Together, these findings identify maternal exercise as a regulator of fetal skeletal development, providing a platform for studying skeletal developmental mechanobiology and suggesting potential therapeutic applications for fetuses with impaired movement.

Clinically, compromised fracture healing often occurs at sites with less muscle coverage and muscle flaps can provide the necessary healing environment for appropriate healing in severe bone loss. However, the underlying mechanisms are largely unknown. Here, we established a mouse reporter model for studying muscle cell contribution to bone fracture repair. Analyzing skeletal muscle scRNA-seq datasets revealed that Prg4 marks a fibro-adipogenic progenitor (FAP) subpopulation. In mice, Prg4+ cells were specifically located in the skeletal muscle, but not at the periosteum or inside cortical bone. These cells expressed FAP markers, responded to muscle injury, and became periosteal cells under normal and muscle injury conditions. Fracture fragmented muscle fibers, rapidly expanded Prg4+ FAPs at the injury site and promoted their migration into the fracture site. Later, they gave rise to many chondrocytes, osteoblasts, and osteocytes in the outer periphery of callus next to muscle. In repaired bones, the descendants of Prg4+ FAPs were detected as mesenchymal progenitors in the periosteum and osteocytes at the prior fracture site. A second fracture activated those cells and stimulated them to become osteoblasts in the inner part of callus. Importantly, ablation of Prg4+ FAPs impaired fracture healing and functional repair. In an intramembranous bone injury model (drill-hole), Prg4+ FAPs became periosteal cells, but their contribution to bone defect repair was significantly less than in fractures. Taken together, we demonstrate the critical role of FAPs in endochondral bone repair and uncover a novel mechanism by which mesenchymal progenitors transform from muscle to cortical bone. Significance statement: Fracture healing is often impaired in areas with less muscle coverage, but the mechanisms behind this are poorly understood. In this work, we uncovered a critical role of specific muscle cells, known as Prg4+ fibro-adipogenic progenitors (FAPs), in bone regeneration. Using mouse models, we show that these muscle-resident mesenchymal progenitors expand after bone injury, migrate to the fracture site, transform into various bone cells in the fracture callus, and eventually become bone surface progenitors after the bone is healed. This finding highlights the importance of muscle cells in endochondral fracture repair and reveals a novel crosstalk mechanism between muscle and bone tissues. Future studies targeting these muscle progenitor cells could develop new treatments for delayed and nonunion fractures.

BACKGROUND: Bones develop to structurally balance strength and mobility. Bone developmental dynamics are influenced by whether an animal is ambulatory at birth. Precocial species, which are ambulatory at birth, develop advanced skeletal maturity in utero and experience postnatal development under mechanical loading. Here, we characterized postnatal bone development in the lower forelimbs of precocial goats using microcomputed tomography and histology. Our analysis focused on the two phalanges 1 (P1) bones and the partially fused metacarpal bone of the goat autopod from birth through adulthood. RESULTS: P1 cortical bone densified rapidly after birth, but cortical thickness increased continually through adulthood. Upon normalization by body mass, the P1 normalized polar moment of inertia was constant over time, suggestive of changes correlating with ambulatory loading. P1 trabecular bone increased in trabecular number and thickness until sexual maturity (12 months), while metacarpal trabeculae grew primarily through trabecular thickening. Unlike prenatal synostosis (i.e., bone fusion) of the metacarpal diaphysis, synostosis of the epiphyses occurred postnatally, prior to growth plate closure, through a unique fibrocartilaginous endochondral ossification. CONCLUSIONS: These findings implicate ambulatory loading in postnatal bone development of precocial goats and identify a novel postnatal synostosis event in the caprine metacarpal epiphysis.

Georgios Kotsaris, Joel D Boerckel; Macrophages: the maestros of bone repair Mechanobiology, Journal of Bone and Mineral Research, , zjaf024, https://doi.o

Compromised vascular supply and insufficient neovascularization impede bone repair, increasing risk of non-union. CYR61, Cysteine-rich angiogenic inducer of 61kD (also known as CCN1), is a matricellular growth factor that has been implicated in fracture repair. Here, we map the distribution of endogenous CYR61 during bone repair and evaluate the effects of recombinant CYR61 delivery on vascularized bone regeneration. In vitro, CYR61 treatment did not alter chondrogenesis or osteogenic gene expression, but significantly enhanced angiogenesis. In a mouse femoral fracture model, CYR61 delivery did not alter cartilage or bone formation, but accelerated neovascularization during fracture repair. Early initiation of ambulatory mechanical loading disrupted CYR61-induced neovascularization. Together, these data indicate that CYR61 delivery can enhance angiogenesis during bone repair, particularly for fractures with stable fixation, and may have therapeutic potential for fractures with limited blood vessel supply.

Abstract Oxygen tension dynamically regulates stem cell fate and tissue regeneration, yet how local oxygen availability is controlled within the bone marrow niche remains poorly understood. While bone marrow injury, such as by bone fracture, disrupts marrow vasculature, the consequences on local oxygen tension remain unclear. Here, we show in mice that while the tissue oxygen tension in bone marrow is low (25 mmHg, ∼4% O 2 ), intracellular oxygenation is heterogeneous and erythroid cells are high in oxygen. Bone fracture elevates oxygen tension in the injured bone marrow (>55 mmHg, ∼8%), which persists for over a week post-injury. This oxygen elevation results not from angiogenesis, but rather from localized expansion of erythroid precursor cells in the injured bone marrow. The activated erythroid precursors synthesize hemoglobin and accumulate oxygen, acting as local modulators of oxygen tension. Blocking transferrin receptor 1 (CD71)–mediated iron uptake impairs hemoglobin synthesis, reduces local oxygen levels, and enhances bone regeneration through increased angiogenesis and osteogenesis. These findings identify erythroid precursors as active regulators of local oxygen availability in the bone marrow niche, which may be targetable to enhance tissue regeneration.

Abstract Bones develop to structurally balance strength and mobility. Bone developmental dynamics are influenced by whether an animal is ambulatory at birth ( i.e., precocial). Precocial species, such as goats, develop advanced skeletal maturity in utero, making them useful models for studying the dynamics of bone formation under mechanical load. Here, we used microcomputed tomography and histology to characterize postnatal bone development in the autopod of the caprine lower forelimb. The caprine autopod features two toes, fused by metacarpal synostosis ( i.e. , bone fusion) prior to birth. Our analysis focused on the phalanges 1 (P1) and metacarpals of the goat autopod from birth through adulthood (3.5 years). P1 cortical bone densified rapidly after birth (half-life using one-phase exponential decay model (τ 1/2 = 1.6 ± 0.4 months), but the P1 cortical thickness increased continually through adulthood (τ 1/2 = 7.2 ± 2.7 mo). Upon normalization by body mass, the normalized polar moment of inertia of P1 cortical bone was constant over time, suggestive of structural load adaptation. P1 trabecular bone increased in trabecular number (τ 1/2 = 6.7 ± 2.8 mo) and thickness (τ 1/2 = 6.6 ± 2.0 mo) until skeletal maturity, while metacarpal trabeculae grew primarily through trabecular thickening (τ 1/2 = 7.9 ± 2.2 mo). Unlike prenatal fusion of the metacarpal diaphysis, synostosis of the epiphyses occurred postnatally, prior to growth plate closure, through a unique fibrocartilaginous endochondral ossification. These findings implicate ambulatory loading in postnatal bone development of precocial goats and identify a novel postnatal synostosis event in the caprine metacarpal epiphysis.

Bone fracture repair initiates by periosteal expansion. The periosteum is typically quiescent, but upon fracture, periosteal cells proliferate and contribute to bone fracture repair. The expansion of the periosteum is regulated by gene transcription; however, the molecular mechanisms behind periosteal expansion are unclear. Here, we show that Yes-Associated Protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) mediate periosteal expansion and periosteal cell proliferation. Bone fracture increases the number of YAP-expressing periosteal cells, and deletion of YAP and TAZ from Osterix (Osx) expressing cells impairs early periosteal expansion. Mechanistically, YAP regulates both 'cell-intrinsic' and 'cell-extrinsic' factors that allow for periosteal expansion. Specifically, we identified Bone Morphogenetic Protein 4 (BMP4) as a cell extrinsic factor regulated by YAP, that rescues the impairment of periosteal expansion upon YAP/TAZ deletion. Together, these data establish YAP mediated transcriptional mechanisms that induce periosteal expansion in the early stages of fracture repair and provide new putative targets for therapeutic interventions.

Abstract Vascular morphogenesis requires persistent endothelial cell motility that is responsive to diverse and dynamic mechanical stimuli. Here, we interrogated the mechanotransductive feedback dynamics that govern endothelial cell motility and vascular morphogenesis. We show that the transcriptional regulators, YAP and TAZ, are activated by mechanical cues to transcriptionally limit cytoskeletal and focal adhesion maturation, forming a conserved mechanotransductive feedback loop that mediates human endothelial cell motility in vitro and zebrafish intersegmental vessel (ISV) morphogenesis in vivo. This feedback loop closes in 4 hours, achieving cytoskeletal equilibrium in 8 hours. Feedback loop inhibition arrested endothelial cell migration in vitro and ISV morphogenesis in vivo. Inhibitor washout at 3 hrs, prior to feedback loop closure, restored vessel growth, but washout at 8 hours, longer than the feedback timescale, did not, establishing lower and upper bounds for feedback kinetics in vivo. Mechanistically, YAP and TAZ induced transcriptional suppression of RhoA signaling to maintain dynamic cytoskeletal equilibria. Together, these data establish the mechanoresponsive dynamics of a transcriptional feedback loop necessary for persistent endothelial cell migration and vascular morphogenesis.

Vascular morphogenesis requires persistent endothelial cell motility that is responsive to diverse and dynamic mechanical stimuli. Here, we interrogated the mechanotransductive feedback dynamics that govern endothelial cell motility and vascular morphogenesis. We show that the transcriptional regulators, YAP and TAZ, are activated by mechanical cues to transcriptionally limit cytoskeletal and focal adhesion maturation, forming a conserved mechanotransductive feedback loop that mediates human endothelial cell motility in vitro and zebrafish intersegmental vessel (ISV) morphogenesis in vivo. This feedback loop closes in 4 hours, achieving cytoskeletal equilibrium in 8 hours. Feedback loop inhibition arrested endothelial cell migration in vitro and ISV morphogenesis in vivo. Inhibitor washout at 3 hrs, prior to feedback loop closure, restored vessel growth, but washout at 8 hours, longer than the feedback timescale, did not, establishing lower and upper bounds for feedback kinetics in vivo. Mechanistically, YAP and TAZ induced transcriptional suppression of RhoA signaling to maintain dynamic cytoskeletal equilibria. Together, these data establish the mechanoresponsive dynamics of a transcriptional feedback loop necessary for persistent endothelial cell migration and vascular morphogenesis.

ABSTRACT Cell-cell fusion is an evolutionarily conserved process that is essential for many functions, including fertilisation and the formation of placenta, muscle and osteoclasts, multinucleated cells that are unique in their ability to resorb bone. The mechanisms of osteoclast multinucleation involve dynamic interactions between the actin cytoskeleton and the plasma membrane that are still poorly characterized. Here, we found that moesin, a cytoskeletal linker protein member of the Ezrin/Radixin/Moesin (ERM) protein family, is activated during osteoclast maturation and plays an instrumental role in both osteoclast fusion and function. In mouse and human osteoclast precursors, moesin inhibition favors their ability to fuse into multinucleated osteoclasts. Accordingly, we demonstrated that moesin depletion decreases membrane-to-cortex attachment and enhances the formation of tunneling nanotubes (TNTs), F-actin-based intercellular bridges that we reveal here to trigger cell-cell fusion. Moesin also controls HIV-1- and inflammation-induced cell fusion. In addition, moesin regulates the formation of the sealing zone, the adhesive structure determining osteoclast bone resorption area, and thus controls bone degradation, via a β3-integrin/RhoA/SLK pathway. Supporting our results, moesin - deficient mice present a reduced density of trabecular bones and increased osteoclast abundance and activity. These findings provide a better understanding of the regulation of cell-cell fusion and osteoclast biology, opening new opportunities to specifically target osteoclast activity in bone disease therapy.

Compromised vascular supply and insufficient neovascularization impede bone repair, increasing risk of non-union. Cyr61, Cysteine-rich angiogenic inducer of 61kD (also known as CCN1), is a matricellular growth factor that is regulated by mechanical cues during fracture repair. Here, we map the distribution of endogenous Cyr61 during bone repair and evaluate the effects of recombinant Cyr61 delivery on vascularized bone regeneration. In vitro, Cyr61 treatment did not alter chondrogenesis or osteogenic gene expression, but significantly enhanced angiogenesis. In a mouse femoral fracture model, Cyr61 delivery did not alter cartilage or bone formation, but accelerated neovascularization during fracture repair. Early initiation of ambulatory mechanical loading disrupted Cyr61-induced neovascularization. Together, these data indicate that Cyr61 delivery can enhance angiogenesis during bone repair, particularly for fractures with stable fixation, and may have therapeutic potential for fractures with limited blood vessel supply.

Skeletal development depends on coordinated angiogenesis and osteogenesis. Bone morphogenetic proteins direct bone formation in part by activating SMAD1/5 signaling in osteoblasts. However, the role of SMAD1/5 in skeletal endothelium is unknown. Here, we found that endothelial cell-conditional SMAD1/5 depletion in juvenile mice caused metaphyseal and diaphyseal hypervascularity, resulting in altered trabecular and cortical bone formation. SMAD1/5 depletion induced excessive sprouting and disrupting the morphology of the metaphyseal vessels, with impaired anastomotic loop formation at the chondro-osseous junction. Endothelial SMAD1/5 depletion impaired growth plate resorption and, upon long-term depletion, abrogated osteoprogenitor recruitment to the primary spongiosa. Finally, in the diaphysis, endothelial SMAD1/5 activity was necessary to maintain the sinusoidal phenotype, with SMAD1/5 depletion inducing formation of large vascular loops and elevated vascular permeability. Together, endothelial SMAD1/5 activity sustains skeletal vascular morphogenesis and function and coordinates growth plate remodeling and osteoprogenitor recruitment dynamics in juvenile mouse bone.

Hydrogels and hydrogel-based materials, thanks to their biocompatibility and biodegradability, are widely used as a supporting matrix for embedding various kinds of luminescent probes for biological sensing applications. Here we describe a family of phosphorescent hydrogels, termed Oxygels, which were designed specifically for local sensing of oxygen by means of Cherenkov-Excited Luminescence Imaging (CELI) in and around tumors during application of radiation therapy. Previously, our group has developed soluble phosphorescent probes, known as Oxyphors, and demonstrated their performance in CELI of oxygen. Oxyphors comprise phosphorescent metalloporphyrins encapsulated inside hydrophobic dendrimers, whose periphery is modified with polyethyleneglycol (PEG) residues. The PEG layer creates a hydrophilic jacket around the dendrimer, precluding interactions of the probe with biomacromolecules. As a result, Oxyphors retain stable calibration parameters, enabling quantitative imaging of oxygen in in vivo. However, locally delivered Oxyphors rapidly diffuse away from the injection sites and spread throughout the body, posing challenges to local oxygen quantification as well as raising concerns in terms of regulatory (FDA) approval. To this end, hydrogel-supported phosphorescent sensors implanted into tissue should allow for continuous local monitoring of oxygen during RT, aiding optimization of treatment protocols and facilitating the development of new types of RT treatment.

Mesenchymal stromal cells (MSCs) are widely used in cell-based therapies and tissue regeneration for their potent secretome, which promotes host cell recruitment and modulates inflammation. Compared to monodisperse cells, MSC spheroids exhibit improved viability and increased secretion of immunomodulatory cytokines. While mechanical stimulation of monodisperse cells can increase cytokine production, the influence of mechanical loading on MSC spheroids is unknown. Here, we evaluated the effect of controlled, uniaxial cyclic compression on the secretion of immunomodulatory cytokines by human MSC spheroids and tested the influence of load-induced gene expression on MSC mechanoresponsiveness. We exposed MSC spheroids, entrapped in alginate hydrogels, to three cyclic compressive regimes with varying stress (L) magnitudes (i.e., 5 and 10 kPa) and hold (H) durations (i.e., 30 and 250 s) L5H30, L10H30, and L10H250. We observed changes in cytokine and chemokine expression dependent on the loading regime, where higher stress regimes tended to result in more exaggerated changes. However, only MSC spheroids exposed to L10H30 induced human THP-1 macrophage polarization toward an M2 phenotype compared to static conditions. Static and L10H30 loading facilitated a strong, interlinked F-actin arrangement, while L5H30 and L10H250 disrupted the structure of actin filaments. This was further examined when the actin cytoskeleton was disrupted via Y-27632. We observed downregulation of YAP-related genes, and the levels of secreted inflammatory cytokines were globally decreased. These findings emphasize the essential role of mechanosignaling in mediating the immunomodulatory potential of MSC spheroids.

Summary: Fetal bone development occurs by conversion of avascular cartilage to vascularized bone at the growth plate. This requires coordinated mobilization of osteoblast precursors with blood vessels. In adult bone, vessel-adjacent osteoblast precursors are maintained by mechanical stimuli; however, the mechanisms by which these cells mobilize and respond to mechanical cues during embryonic development are unknown. Here, we show that the mechanoresponsive transcriptional regulators YAP and TAZ spatially couple osteoblast precursor mobilization to angiogenesis, regulate vascular morphogenesis to control cartilage remodeling, and mediate mechanoregulation of embryonic murine osteogenesis. Mechanistically, YAP and TAZ regulate a subset of osteoblast-lineage cells, identified by single-cell RNA sequencing as vessel-associated osteoblast precursors, which regulate transcriptional programs that direct blood vessel invasion through collagen-integrin interactions and Cxcl12. Functionally, in 3D human cell co-culture, CXCL12 treatment rescued angiogenesis impaired by stromal cell YAP/TAZ depletion. Together, these data establish functions of the vessel-associated osteoblast precursors in bone development.

Insufficient bone fracture repair represents a major clinical and societal burden and novel strategies are needed to address it. Our data reveal that the transforming growth factor-β superfamily member Activin A became very abundant during mouse and human bone fracture healing but was minimally detectable in intact bones. Single-cell RNA-sequencing revealed that the Activin A-encoding gene Inhba was highly expressed in a unique, highly proliferative progenitor cell (PPC) population with a myofibroblast character that quickly emerged after fracture and represented the center of a developmental trajectory bifurcation producing cartilage and bone cells within callus. Systemic administration of neutralizing Activin A antibody inhibited bone healing. In contrast, a single recombinant Activin A implantation at fracture site in young and aged mice boosted: PPC numbers; phosphorylated SMAD2 signaling levels; and bone repair and mechanical properties in endochondral and intramembranous healing models. Activin A directly stimulated myofibroblastic differentiation, chondrogenesis and osteogenesis in periosteal mesenchymal progenitor culture. Our data identify a distinct population of Activin A-expressing PPCs central to fracture healing and establish Activin A as a potential new therapeutic tool.

Mechanobiology is an interdisciplinary field that aims to understand how physical forces impact biological systems. Enhancing our knowledge of mechanobiology has become increasingly important for understanding human disease and developing novel therapeutics. There is a societal need to teach diverse students principles of mechanobiology so that we may collectively expand our knowledge of this subject and apply new principles to improving human health. Toward this goal, we designed, implemented, and evaluated a hands-on, inquiry-based learning (IBL) module to teach students principles of cell-biomaterial interactions. This module was designed to be hosted in two 3-h sessions, over two consecutive days. During this time, students learned how to synthesize and mechanically test biomaterials, culture bacteria cells, and assess effects of matrix stiffness on bacteria cell proliferation. Among the 73 students who registered to participate in our IBL mechanobiology module, 40 students completed both days and participated in this study. A vast majority of the participants were considered underrepresented minority (URM) students based on race/ethnicity. Using pre/post-tests, we found that students experienced significant learning gains of 33 percentage points from completing our IBL mechanobiology module. In addition to gaining knowledge of mechanobiology, validated pre/post-surveys showed that students also experienced significant improvements in scientific literacy. Instructors may use this module as described, increase the complexity for an undergraduate classroom assignment, or make the module less complex for K-12 outreach. As presented, this IBL mechanobiology module effectively teaches diverse students principles of mechanobiology and scientific inquiry. Deploying this module, and similar IBL modules, may help advance the next generation of mechanobiologists.

Abstract Vascular morphogenesis requires persistent endothelial cell motility that is responsive to diverse and dynamic mechanical stimuli. Here, we interrogated the mechanotransductive feedback dynamics that govern endothelial cell motility and vascular morphogenesis. We show that the transcriptional regulators, YAP and TAZ, are activated by mechanical cues to transcriptionally limit cytoskeletal and focal adhesion maturation, forming a conserved mechanotransductive feedback loop that mediates human endothelial cell motility in vitro and zebrafish intersegmental vessel (ISV) morphogenesis in vivo. This feedback loop closes in 4 hours, achieving cytoskeletal equilibrium in 8 hours. Feedback loop inhibition arrested endothelial cell migration in vitro and ISV morphogenesis in vivo. Inhibitor washout at 3 hrs, prior to feedback loop closure, restored vessel growth, but washout at 8 hours, longer than the feedback timescale, did not, establishing lower and upper bounds for feedback kinetics in vivo. Mechanistically, YAP and TAZ induced transcriptional suppression of myosin II activity to maintain dynamic cytoskeletal equilibria. Together, these data establish the mechanoresponsive dynamics of a transcriptional feedback loop necessary for persistent endothelial cell migration and vascular morphogenesis.

Vascular morphogenesis requires persistent endothelial cell motility that is responsive to diverse and dynamic mechanical stimuli. Here, we interrogated the mechanotransductive feedback dynamics that govern endothelial cell motility and vascular morphogenesis. We show that the transcriptional regulators, YAP and TAZ, are activated by mechanical cues to transcriptionally limit cytoskeletal and focal adhesion maturation, forming a conserved mechanotransductive feedback loop that mediates human endothelial cell motility in vitro and zebrafish intersegmental vessel (ISV) morphogenesis in vivo. This feedback loop closes in 4 hours, achieving cytoskeletal equilibrium in 8 hours. Feedback loop inhibition arrested endothelial cell migration in vitro and ISV morphogenesis in vivo. Inhibitor washout at 3 hrs, prior to feedback loop closure, restored vessel growth, but washout at 8 hours, longer than the feedback timescale, did not, establishing lower and upper bounds for feedback kinetics in vivo. Mechanistically, YAP and TAZ induced transcriptional suppression of RhoA signaling to maintain dynamic cytoskeletal equilibria. Together, these data establish the mechanoresponsive dynamics of a transcriptional feedback loop necessary for persistent endothelial cell migration and vascular morphogenesis.