Biology subjects

Boerckel, J. D.

Publications and source records attributed to Boerckel, J. D..

At least 19 recordsLinked to original sources

bioRxiv2026

Impaired loading following denervation elicits dynamic, differential growth trajectories of musculoskeletal tissues

Physical forces dictate the function and maintenance of adult musculoskeletal tissues. Yet, the impact of biophysical cues on the growth and maturation of these tissues, especially dense connective tissues, remains unclear. Here, we investigated the effects of neonatal mechanical loading on early postnatal growth of the musculoskeletal tissues of the lower limb, with particular focus on the Achilles tendon. We performed sciatic nerve resection (SNR) on one-day-old mice to denervate the hamstrings and distal hindlimb muscles. Sciatic Nerve Resection resulted in severe and sustained alterations in gait, accompanied by an altered transcriptional landscape in distal musculoskeletal tissues, with marked reductions in bone and tendon growth and smaller, fibrotic hindlimb musculature. Sciatic nerve resection impaired cumulative growth in the Achilles tendon, shifting cell proliferation and matrix-driven growth trajectories and altering mechanotransduction gene programs toward inflammation and matrix catabolism with age. This work highlights the essential role of mechanical loading in driving mechanosignaling and postnatal growth trajectories in musculoskeletal tissues of the developing limb, with implications for overall musculoskeletal health in adulthood.

bioengineering↗

bioRxiv2026

Mechano-activated synovial cells instigate OA-like disease progression in the absence of joint injury

While altered tissue mechanics and cellular mechanoactivation accompany osteoarthritis, whether dysregulated mechanotransduction can initiate joint disease remains unclear. Here, we used intra-articular delivery of cell-penetrating TAT-Cre recombinase to locally perturb the actomyosin-Hippo mechanotransduction axis in mice. Deletion of Lats1/2, the negative regulators of the mechanosensitive transcriptional coactivators YAP and TAZ, induced sustained joint hyperalgesia, altered gait, synovial hyperplasia and fibrosis, vascular remodeling, and articular cartilage degeneration in the absence of a destabilizing injury. Single-cell transcriptomics revealed the emergence of Cthrc1+ matrix-remodeling fibroblasts and accumulation of Mrc1+Trem2+ macrophages during this pathological process. Histological analyses showed persistent contractile stromal remodeling and an early macrophage response that partially subsided over four weeks. Conversely, local deletion of Myh9/10, encoding the force-generating heavy chains of non-muscle myosin II, reduced hyperalgesia, synovial lining hyperplasia, alpha-SMA+ cell accumulation, and macrophage infiltration during post-traumatic osteoarthritis (PTOA) pathogenesis. However, this intervention did not detectably reduce cartilage degeneration at eight weeks. These findings establish synovial mechanotransduction as a causal driver of joint disease via immune-stromal remodeling and pain development. In addition, partial suppression of contractility separates symptomatic from structural outcomes in PTOA. Synovial mechanotransduction therefore represents a target for modulating pathological stromal-immune remodeling and joint pain.

pathology↗

bioRxiv2026

Cytoskeletal disassembly by optogenetic control of RhoA signaling termination

Cellular morphodynamics require adaptive cytoskeletal remodeling, mediated by precisely coordinated activation and termination of RhoA GTPase signaling. RhoA activation is well-studied, but the kinetics and molecular basis of signaling termination remain poorly understood. We engineered an optogenetic toolbox on the single-component BcLOV4 platform for bidirectional control of RhoA activation (opto-GEF11) and termination (opto-DLC1). Prior studies have inferred GTPase inactivation kinetics indirectly, by tracking passive recovery from an activated state, but whether this reflects the kinetics of forward signaling termination remains unclear. Using opto-DLC1, we show that direct RhoA termination is an order of magnitude faster than passive disactivation, despite comparable signaling amplitude. Mechanistically, opto-DLC1 triggered rapid actin disassembly through cofilin disinhibition but drove YAP nuclear efflux at half the rate of opto-GEF11-induced nuclear influx. Together, these findings introduce a platform technology for controlling protein signaling termination, resolve RhoA activation and termination kinetics with sub-second precision, and reveal a mechanistic asymmetry between signaling activation and termination that enables cytoskeletal homeostasis.

bioengineering↗

bioRxiv2026

Osteocyte Perilacunar/canalicular Remodeling (PLR) Drives Spatially Heterogeneous Lacunar Remodeling During and After Lactation

BackgroundPregnancy 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/HypothesisThis 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. MethodsFemale wild-type (WT) and osteocyte-specific PTH/PTHrP receptor knockout (cKO) mice were studied across reproductive stages. Longitudinal in vivo DXA and CT 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). ResultsAt 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. ConclusionOsteocyte 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.

bioengineering↗

bioRxiv2026

Degeneration-Inspired Architectural States Defined by Voronoi Point Spacing and Surface-Mediated Rescue of Osteogenic Dysfunction in 3D-Printed Scaffolds

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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/725650v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@15b1204org.highwire.dtl.DTLVardef@1fbdce6org.highwire.dtl.DTLVardef@638215org.highwire.dtl.DTLVardef@f1370d_HPS_FORMAT_FIGEXP M_FIG C_FIG Statement of SignificanceMost 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.

bioengineering↗

bioRxiv2026

Plasma-Enabled Multiscale Coupling of Architecture and Biointerfaces Drives Osteogenesis in 3D-Printed Gyroid Scaffolds

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 GraphicsA 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/718992v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1d29685org.highwire.dtl.DTLVardef@983752org.highwire.dtl.DTLVardef@15816f5org.highwire.dtl.DTLVardef@4b4f50_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

CRISPR-mediated conditional mutagenesis of Smad1/5/8 reveals BMP/GDF signaling restricts postnatal bone overgrowth

The BMP/GDF branch of TGF-{beta} 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.

genetics↗

bioRxiv2025

Biphasic Mechanical Loading Disrupts Cytoskeletal Symmetry in 3D Architected Scaffolds

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 StatementCells 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.

bioengineering↗

bioRxiv2025

Maternal exercise rescues fetal akinesia-impaired joint and bone development

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 (Spd) 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 Spd 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.

developmental biology↗

bioRxiv2025

Prg4+ fibro-adipogenic progenitors in muscle are crucial for bone fracture repair

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 statementFracture 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.

cell biology↗

bioRxiv2025

Local erythropoiesis directs oxygen availability in bone fracture repair

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% O2), 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.

cell biology↗

bioRxiv2024

Dynamics of postnatal bone development and epiphyseal synostosis in the caprine autopod

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 ({tau}1/2 = 1.6 {+/-} 0.4 months), but the P1 cortical thickness increased continually through adulthood ({tau}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 ({tau}1/2 = 6.7 {+/-} 2.8 mo) and thickness ({tau}1/2 = 6.6 {+/-} 2.0 mo) until skeletal maturity, while metacarpal trabeculae grew primarily through trabecular thickening ({tau}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.

developmental biology↗

bioRxiv2024

YAP regulates periosteal expansion in fracture repair

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 co-activator 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.

cell biology↗

bioRxiv2024

Moesin activation controls bone resorption and tunneling nanotube-dependent osteoclast fusion

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 {beta}3-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.

cell biology↗

bioRxiv2024

Cyr61 delivery promotes angiogenesis during bone fracture repair

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.

bioengineering↗

bioRxiv2023

YAP and TAZ couple osteoblast precursor mobilization to angiogenesis and mechanoregulated bone development

Endochondral ossification requires coordinated mobilization of osteoblast precursors with blood vessels. During adult bone homeostasis, vessel adjacent osteoblast precursors respond to and are maintained by mechanical stimuli; however, the mechanisms by which these cells mobilize and respond to mechanical cues during embryonic development are unknown. Previously, we found that deletion of the mechanoresponsive transcriptional regulators, YAP and TAZ, from Osterix-expressing osteoblast precursors and their progeny caused perinatal lethality. Here, we show that embryonic YAP/TAZ signaling couples vessel-associated osteoblast precursor mobilization to angiogenesis in developing long bones. Osterix-conditional YAP/TAZ deletion impaired endochondral ossification in the primary ossification center but not intramembranous osteogenesis in the bone collar. Single-cell RNA sequencing revealed YAP/TAZ regulation of the angiogenic chemokine, Cxcl12, which was expressed uniquely in vessel-associated osteoblast precursors. YAP/TAZ signaling spatially coupled osteoblast precursors to blood vessels and regulated vascular morphogenesis and vessel barrier function. Further, YAP/TAZ signaling regulated vascular loop morphogenesis at the chondro-osseous junction to control hypertrophic growth plate remodeling. In human cells, mesenchymal stromal cell co-culture promoted 3D vascular network formation, which was impaired by stromal cell YAP/TAZ depletion, but rescued by recombinant CXCL12 treatment. Lastly, YAP and TAZ mediated mechanotransduction for load-induced osteogenesis in embryonic bone.

developmental biology↗

bioRxiv2023

Endothelial SMAD1/5 signaling couples angiogenesis to osteogenesis during long bone growth

Skeletal development depends on coordinated angiogenesis and osteogenesis. Bone morphogenetic proteins direct bone development 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 cancellous and cortical bone formation. SMAD1/5 depletion induced excessive sprouting, disrupting the columnar structure of the metaphyseal vessels and impaired anastomotic loop morphogenesis 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, featuring elevated endomucin expression, ectopic tip cell formation, and hyperpermeability. Together, endothelial SMAD1/5 activity sustains skeletal vascular morphogenesis and function and coordinates growth plate remodeling and osteoprogenitor recruitment dynamics during bone growth.

developmental biology↗

bioRxiv2022

Activin A promotes bone fracture repair and acts through a novel myofibroblastic cell population in callus

Insufficient bone fracture repair represents a significant clinical burden, and identification of novel therapeutics enhancing repair would have substantial clinical and societal impact. Activin A is a TGF-{beta} protein superfamily member known to stimulate ectopic bone formation, but its roles in fracture repair and its therapeutic potentials remain unclear. Using two mouse tibia fracture repair models, here we mapped activin A expression at the tissue and single cell levels, tested its requirement for normal repair and evaluated its ability to enhance repair when provided exogenously. Activin A was minimally expressed in periosteum of intact bones but was markedly upregulated in developing callus soon after fracture. Single cell RNA-sequencing revealed that the activin A-encoding gene Inhba marked a unique, highly proliferative progenitor cell (PPC) population with a myofibroblast character which emerged over repair time and lay at the center of a developmental trajectory bifurcation producing cartilage and bone cells within callus. Systemic administration of a neutralizing activin A antibody impaired fracture repair and its endochondral and intramembranous phases, whereas local delivery of recombinant activin A enhanced repair. Activin A delivery also induced SMAD2 phosphorylation in vivo and increased the fraction of SMA+ myofibroblasts within fracture callus. Gain- and loss-of-function experiments in vitro showed that activin A directly stimulated myofibroblast differentiation, chondrogenesis and osteogenesis in periosteal progenitor cells. Together, these data identify a unique population of Inhba-expressing proliferative progenitor cells that give rise to chondrocytes and osteoblasts during fracture healing and establish activin A as a potential new therapeutic tool to enhance it. One Sentence SummaryDeficits in bone fracture repair remain a clinical challenge and the present study provides evidence for the therapeutic potentials of activin A

pathology↗