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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.

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. ispartof: bioRxiv ispartof: location:United States status: Published online

The skin has a pronounced ability to adapt to physical changes in the environment by exhibiting plasticity at the cellular level. Transient mechanical deformations applied to the skin are accommodated without permanent changes to tissue structure. However, sustained physical stress induces long-lasting alterations in the skin, which are mediated by shifts in the fates of epidermal stem cells. To investigate this phenomenon, we implemented two-photon intravital imaging to capture the responses of epidermal cells when an acute mechanical force is applied to the live skin. We show that mechanical stress induces the formation of intracellular vesicles in epidermal stem cells, which are filled with extracellular fluid and gradually enlarge, causing the deformation of the cell nucleus. By lineage tracing analysis we demonstrate that the degree of nuclear deformation is linked to cell fate. Utilizing a fluorescent in vivo reporter, to capture intracellular calcium dynamics, we show that mechanical force induces a sustained increase in intracellular calcium within basal epidermal stem cells. Conditional deletion of Piezo1, a mechanosensitive ion channel, alters intracellular calcium dynamics and increases the number of stress vesicles in epidermal stem cells. Using a human skin xenograft model, we show that stress vesicles are a conserved phenomenon in mammalian skin. This study uncovers stress vesicles as key manifestations of the mechanism that regulates the fate of epidermal stem cells under conditions of mechanical stress, in which Piezo1 and calcium dynamics are also involved.

The journals of the American Society for Bone and Mineral Research (the Journal of Bone and Mineral Research [JBMR] and its sister journal JBMR Plus) recognize peer review, whether pre- or post-publication, as an essential guard of scientific integrity and rigor that shapes academic discourse in our field. In this Perspective, we present a vision and philosophy of peer review in a rapidly changing publishing landscape. We emphasize the importance of journal peer reviewers as active players in shaping collegial behavior in the musculoskeletal research community and provide information about benefits and resources available for reviewers and reviewers-in-training. Publishing is becoming increasingly transparent, bringing benefits to authors, to reviewers, and to the scientific community at large. We discuss new initiatives such as transparent peer review and preprint servers, the ways they are changing scientific publishing, and how JBMR is responding to broaden the impact of musculoskeletal research. We emphasize the need to change any perception of peer reviewers as gatekeepers to viewing them as shepherds, who partner with authors and editors in the publishing endeavor. Promoting access, transparency, and collegiality in the way we assess science in our community will elevate its quality, clarify its communication, and increase its societal impact. © 2021 American Society for Bone and Mineral Research (ASBMR).

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 motilityin vitroand zebrafish intersegmental vessel (ISV) morphogenesisin vivo. This feedback loop closes in 4 hours, achieving cytoskeletal equilibrium in 8 hours. Feedback loop inhibition arrested endothelial cell migrationin vitroand ISV morphogenesisin 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 kineticsin 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.

Insufficient blood vessel supply is a primary limiting factor for regenerative approaches to large bone defect repair. Recombinant bone morphogenetic protein-2 (BMP-2) delivery induces robust bone formation and has been observed to enhance neovascularization, but whether the angiogenic effects of BMP-2 are due to direct endothelial cell stimulation or due to indirect paracrine signaling remain unclear. In this study, we evaluated the effects of BMP-2 delivery on vascularized bone regeneration and tested whether BMP-2 induces neovascularization directly or indirectly. We found that delivery of BMP-2 (5 μg) enhanced both bone formation and neovascularization in critically sized (8 mm) rat femoral bone defects; however, BMP-2 did not directly stimulate angiogenesis in vitro. In contrast, conditioned medium from both mesenchymal progenitor cells and osteoblasts induced endothelial cell migration in vitro, suggesting a paracrine mechanism of BMP-2 action. Consistent with this inference, codelivery of BMP-2 with endothelial colony forming cells to a heterotopic site, distant from the skeletal stem cell-rich bone marrow niche, induced ossification but had no effect on neovascularization. Taken together, these data suggest that paracrine activation of osteoprogenitor cells is an important contributor to neovascularization during BMP-2-mediated bone regeneration. IMPACT STATEMENT: In this study, we show that bone morphogenetic protein-2 (BMP-2) robustly induces neovascularization during tissue-engineered large bone defect regeneration, and we found that BMP-2 induced angiogenesis, in part, through paracrine signaling from osteoprogenitor cells.

The importance of transcription during cell motility is controversial. Mason et al. show that YAP/TAZ-mediated transcription is required to limit cytoskeletal tension generation and permit persistent cell motility. This pathway is defined as a negative feedback loop whereby Rho-ROCK-myosin activate YAP and TAZ, which limit myosin activation through NUAK2 expression., Cell migration initiates by traction generation through reciprocal actomyosin tension and focal adhesion reinforcement, but continued motility requires adaptive cytoskeletal remodeling and adhesion release. Here, we asked whether de novo gene expression contributes to this cytoskeletal feedback. We found that global inhibition of transcription or translation does not impair initial cell polarization or migration initiation, but causes eventual migratory arrest through excessive cytoskeletal tension and over-maturation of focal adhesions, tethering cells to their matrix. The transcriptional coactivators YAP and TAZ mediate this feedback response, modulating cell mechanics by limiting cytoskeletal and focal adhesion maturation to enable persistent cell motility and 3D vasculogenesis. Motile arrest after YAP/TAZ ablation was partially rescued by depletion of the YAP/TAZ-dependent myosin phosphatase regulator, NUAK2, or by inhibition of Rho-ROCK-myosin II. Together, these data establish a transcriptional feedback axis necessary to maintain a responsive cytoskeletal equilibrium and persistent migration.

Identifying chemical regulators of biological pathways is currently a time-consuming bottleneck in developing therapeutics and small-molecule research tools. Typically, thousands to millions of candidate small molecules are tested in target-based biochemical screens or phenotypic cell-based screens, both expensive experiments customized to a disease of interest. Here, we instead use a broad, virtual screening approach that matches compounds to pathways based on phenotypic information in public data. Our computational strategy efficiently uncovered small molecule regulators of three pathways, containing p38ɑ (MAPK14), YAP1, or PPARGC1A (PGC-1α). We first selected genes whose overexpression yielded distinct image-based profiles in the Cell Painting assay, a microscopy assay involving six stains that label eight cellular organelles/components. To identify small molecule regulators of pathways involving those genes, we used publicly available Cell Painting profiles of 30,616 small molecules to identify compounds that yield morphological effects either positively or negatively correlated with image-based profiles for specific genes. Subsequent assays validated compounds that impacted the predicted pathway activities. This image profile-based drug discovery approach could transform both basic research and drug discovery by identifying useful compounds that modify pathways of biological and therapeutic interest, thus using a computational query to replace certain customized labor- and resource-intensive screens.

Mechanotransduction describes activation of gene expression by changes in the cell’s physical microenvironment. Recent experiments show that mechanotransduction can lead to long-term “mechanical memory,” in which cells cultured on stiff substrates for sufficient time (priming phase) maintain altered phenotype after switching to soft substrates (dissipation phase) as compared to unprimed controls. The timescale of memory acquisition and retention is orders of magnitude larger than the timescale of mechanosensitive cellular signaling, and memory retention time changes continuously with priming time. We develop a model that captures these features by accounting for positive reinforcement in mechanical signaling. The sensitivity of reinforcement represents the dynamic transcriptional state of the cell composed of protein lifetimes and three-dimensional chromatin organization. Our model provides a single framework connecting microenvironment mechanical history to cellular outcomes ranging from no memory to terminal differentiation. Predicting cellular memory of environmental changes can help engineer cellular dynamics through changes in culture environments.

Mechanotransduction describes activation of gene expression by changes in the cell’s physical microenvironment. Recent experiments show that mechanotransduction can lead to long-term “mechanical memory”, where cells cultured on stiff substrates for sufficient time (priming phase) maintain altered phenotype after switching to soft substrates (dissipation phase), as compared to unprimed controls. The timescale of memory acquisition and retention is orders of magnitude larger than the timescale of mechanosensitive cellular signaling, and memory retention time changes continuously with priming time. We develop a model that captures these features by accounting for positive reinforcement in mechanical signaling. The sensitivity of reinforcement represents the dynamic transcriptional state of the cell composed of protein lifetimes and 3D chromatin organization. Our model provides a single framework connecting microenvironment mechanical history to cellular outcomes ranging from no memory to terminal differentiation. Predicting cellular memory of environmental changes can help engineer cellular dynamics through changes in culture environments.

We created optogenetic tools to control RhoA GTPase, a central regulator of actin organization and actomyosin contractility. RhoA GTPase, or its upstream activating GEF effectors, were fused to BcLOV4, a photoreceptor that can be dynamically recruited to the plasma membrane by a light-regulated protein-lipid electrostatic interaction with the inner leaflet. Direct membrane recruitment of these effectors induced potent contractile signaling sufficient to separate adherens junctions in response to as little as one pulse of blue light. Cytoskeletal morphology changes were dependent on the alignment of the spatially patterned stimulation with the underlying cell polarization. RhoA-mediated cytoskeletal activation induced YAP nuclear localization within minutes and subsequent mechanotransduction, verified by YAP- TEAD transcriptional activity. These single-component tools, which do not require protein binding partners, offer spatiotemporally precise control over RhoA signaling that will advance the study of its diverse regulatory roles in cell migration, morphogenesis, and cell cycle maintenance.

Biomimetic bone tissue engineering strategies partially recapitulate development. We recently showed functional restoration of femoral defects using scaffold-free human mesenchymal stem cell (hMSC) condensates featuring localized morphogen presentation with delayed in vivo mechanical loading. Possible effects of construct geometry on healing outcome remain unclear. Here, we hypothesized that localized presentation of transforming growth factor (TGF)-β1 and bone morphogenetic protein (BMP)-2 to engineered hMSC tubes mimicking femoral diaphyses induces endochondral ossification, and that TGF-β1 + BMP-2-presenting hMSC tubes enhance defect healing with delayed in vivo loading vs. loosely packed hMSC sheets. Localized morphogen presentation stimulated chondrogenic priming/endochondral differentiation in vitro. Subcutaneously, hMSC tubes formed cartilage templates that underwent bony remodeling. Orthotopically, hMSC tubes stimulated more robust endochondral defect healing vs. hMSC sheets. Tissue resembling normal growth plate was observed with negligible ectopic bone. This study demonstrates interactions between hMSC condensation geometry, morphogen bioavailability, and mechanical cues to recapitulate development for biomimetic bone tissue engineering., Herberg et al. previously showed functional healing of femoral defects using scaffold-free human mesenchymal stem cell (hMSC) condensates with localized morphogen presentation. In this study, they report the importance of the tubular geometry of MSC condensates in long bone regeneration. Unlike loosely packed hMSC sheets, only hMSC tubes induced regenerate tissue partially resembling normal growth plate.

PURPOSE OF REVIEW: The development of the skeleton is controlled by cellular decisions determined by the coordinated activation of multiple transcription factors. Recent evidence suggests that the transcriptional regulator proteins, Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), could have important roles in directing the activity of these transcriptional programs. However, in vitro evidence for the roles of YAP and TAZ in skeletal cells has been hopelessly contradictory. The goal of this review is to provide a cross-sectional view on the state of the field and to synthesize the available data toward a unified perspective. RECENT FINDINGS: YAP and TAZ are regulated by diverse upstream signals and interact downstream with multiple transcription factors involved in skeletal development, positioning YAP and TAZ as important signal integration nodes in an hourglass-shaped signaling pathway. Here, we provide a survey of putative transcriptional co-effectors for YAP and TAZ in skeletal cells. Synthesizing the in vitro data, we conclude that TAZ is consistently pro-osteogenic in function, while YAP can exhibit either pro- or anti-osteogenic activity depending on cell type and context. Synthesizing the in vivo data, we conclude that YAP and TAZ combinatorially promote developmental bone formation, bone matrix homeostasis, and endochondral fracture repair by regulating a variety of transcriptional programs depending on developmental stage. SUMMARY: Here, we discuss the current understanding of the roles of the transcriptional regulators, YAP and TAZ in skeletal development, and provide recommendations for continued study of molecular mechanisms, mechanotransduction, and therapeutic implications for skeletal disease.

Bone development and repair occur by endochondral ossification of a cartilage anlage, or template. Endochondral ossification is regulated by mechanical cues. Recently, we found that in vivo mechanical loading promoted regeneration of large bone defects through endochondral ossification, in a manner dependent on the timing of load initiation. Here, we have developed an in vitro model of the cartilage anlage to test whether the chondrogenic differentiation state alters the response to dynamic mechanical compression. We cultured human bone marrow stromal cells (hMSCs) at high cell density in fibrin hydrogels under chondrogenic priming conditions for periods of 0, 2, 4, or 6 weeks prior to two weeks of dynamic mechanical loading. Samples were evaluated by biomechanical testing, biochemical analysis of collagen and glycosaminoglycan (GAG) deposition, gene expression analysis, and immunohistological analysis, in comparison to time-matched controls cultured under static conditions. We found that dynamic loading increased the mechanical stiffness of engineered anlagen in a manner dependent on the duration of chondrogenic priming prior to load initiation. For chondrogenic priming times of 2 weeks or greater, dynamic loading enhanced the expression of type II collagen and aggrecan, although no significant changes in overall levels of matrix deposition was observed. For priming periods less than 4 weeks, dynamic loading generally supressed markers of hypertrophy and osteogenesis, although this was not observed if the priming period was extended to 6 weeks, where loading instead enhanced the expression of type X collagen. Taken together, these data demonstrate that the duration of chondrogenic priming regulates the endochondral response to dynamic mechanical compression in vitro, which may contribute to the effects of mechanical loading on endochondral bone development, repair, and regeneration in vivo.

Extracellular matrix deformation temporally regulates angiogenesis and upstream angiogenic and mechanosensitive pathways., Mechanical cues influence tissue regeneration, and although vasculature is known to be mechanically sensitive, little is known about the effects of bulk extracellular matrix deformation on the nascent vessel networks found in healing tissues. Previously, we found that dynamic matrix compression in vivo potently regulated revascularization during bone tissue regeneration; however, whether matrix deformations directly regulate angiogenesis remained unknown. Here, we demonstrated that load initiation time, magnitude, and mode all regulate microvascular growth, as well as upstream angiogenic and mechanotransduction signaling pathways. Immediate load initiation inhibited angiogenesis and expression of early sprout tip cell selection genes, while delayed loading enhanced microvascular network formation and upstream signaling pathways. This research provides foundational understanding of how extracellular matrix mechanics regulate angiogenesis and has critical implications for clinical translation of new regenerative medicine therapies and physical rehabilitation strategies designed to enhance revascularization during tissue regeneration.

In response to bone fracture, periosteal progenitor cells proliferate, expand, and differentiate to form cartilage and bone in the fracture callus. These cellular functions require the coordinated activation of multiple transcriptional programs, and the transcriptional regulators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) regulate osteochondroprogenitor activation during endochondral bone development. However, recent observations raise important distinctions between the signaling mechanisms used to control bone morphogenesis and repair. Here, we tested the hypothesis that YAP and TAZ regulate osteochondroprogenitor activation during endochondral bone fracture healing. Constitutive YAP and/or TAZ deletion from Osterix-expressing cells impaired both cartilage callus formation and subsequent mineralization. However, this could be explained either by direct defects in osteochondroprogenitor differentiation after fracture, or by developmental deficiencies in the progenitor cell pool prior to fracture. Consistent with the second possibility, we found that developmental YAP/TAZ deletion produced long bones with impaired periosteal thickness and cellularity. Therefore, to remove the contributions of developmental history, we next generated adult onset-inducible knockout mice (using Osx1-CretetOff) in which YAP and TAZ were deleted prior to fracture, but after normal development. Adult onset-induced YAP/TAZ deletion had no effect on cartilaginous callus formation, but impaired bone formation at 14 days post-fracture (dpf). Earlier, at 4 dpf, adult onset-induced YAP/TAZ deletion impaired the proliferation and expansion of osteoblast precursor cells located in the shoulder of the callus. Further, activated periosteal cells isolated from this region at 4 dpf exhibited impaired osteogenic differentiation in vitro upon YAP/TAZ deletion. Finally, confirming the effects on osteoblast function in vivo, adult onset-induced YAP/TAZ deletion impaired bone formation in the callus shoulder at 7 dpf, prior to the initiation of endochondral ossification. Together, these data show that YAP and TAZ promote the expansion and differentiation of periosteal osteoblast precursors to accelerate bone fracture healing.

Osteocytes are dynamic, bone matrix-remodeling cells that form an intricate network of interconnected projections through the bone matrix, called the lacunar-canalicular system. Osteocytes are the dominant mechanosensory cells in bone and their mechanosensory and mechanotransductive functions follow their morphological form. During osteocytogenesis and development of the osteocyte lacunar-canalicular network, osteocytes must dramatically remodel both their cytoskeleton and their extracellular matrix. In this review, we summarize our current understanding of the mechanisms that govern osteocyte differentiation, cytoskeletal morphogenesis, mechanotransduction, and matrix remodeling. We postulate that the physiologic activation of matrix remodeling in adult osteocytes, known as perilacunar/canalicular remodeling (PLR) represents a re-activation of the developmental program by which the osteocyte network is first established. While much of osteocyte biology remains unclear, new tools and approaches make the present moment a particularly fruitful and exciting time to study the development of these remarkable cells.

Chondrocyte maturation during cartilage development occurs under diverse and dynamic mechanical environments. Mechanical stimulation through bioreactor culture may mimic these conditions to direct cartilage tissue engineering in vitro. Mechanical cues can promote chondrocyte homeostasis or hypertrophy and mineralization, depending potentially on the timing of load application. Here, we tested the effects of chondrogenic priming duration on the response of engineered human cartilage constructs to dynamic mechanical compression. We cultured human bone marrow stromal cells (hMSCs) in fibrin hydrogels under chondrogenic priming conditions for periods of 0, 2, 4, or 6 weeks prior to two weeks of either static culture or dynamic compression. We measured construct mechanical properties, cartilage matrix composition, and gene expression. Dynamic compression increased the equilibrium and dynamic modulus of the engineered tissue, depending on the duration of chondrogenic priming. For priming times of 2 weeks or greater, dynamic compression enhanced COL2A1 and AGGRECAN mRNA expression at the end of the loading period, but did not alter total collagen or glycosaminoglycan matrix deposition. Load initiation at priming times of 4 weeks or less repressed transient osteogenic signaling (RUNX2, OPN) and expression of CYR61, a YAP/TAZ-TEAD-target gene. No suppression of osteogenic gene expression was observed if loading was initiated after 6 weeks of in vitro priming, when mechanical stimulation was observed to increase the expression of type X collagen. Taken together, these data demonstrate that the duration of in vitro chondrogenic priming regulates the cell response to dynamic mechanical compression and suggests that early loading may preserve chondrocyte homeostasis while delayed loading may support cartilage maturation.

The fields of developmental biology and tissue engineering have been revolutionized in recent years by technological advancements, expanded understanding, and biomaterials design, leading to the emerging paradigm of "developmental" or "biomimetic" tissue engineering. While developmental biology and tissue engineering have long overlapping histories, the fields have largely diverged in recent years at the same time that crosstalk opportunities for mutual benefit are more salient than ever. In this perspective article, we will use musculoskeletal development and tissue engineering as a platform on which to discuss these emerging crosstalk opportunities and will present our opinions on the bright future of these overlapping spheres of influence. The multicellular programs that control musculoskeletal development are rapidly becoming clarified, represented by shifting paradigms in our understanding of cellular function, identity, and lineage specification during development. Simultaneously, advancements in bioartificial matrices that replicate the biochemical, microstructural, and mechanical properties of developing tissues present new tools and approaches for recapitulating development in tissue engineering. Here, we introduce concepts and experimental approaches in musculoskeletal developmental biology and biomaterials design and discuss applications in tissue engineering as well as opportunities for tissue engineering approaches to inform our understanding of fundamental biology. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2356-2368, 2017.

Pedicle screw loosening has been implicated in recurrent back pain after lumbar spinal fusion, but the degree of loosening has not been systematically quantified in patients. Instrumentation removal is an option for patients with successful arthrodesis, but remains controversial. Here, we quantified pedicle screw loosening by measuring screw insertion and/or removal torque at high statistical power (beta = 0.02) in N = 108 patients who experienced pain recurrence despite successful fusion after posterior instrumented lumbar fusion with anterior lumbar interbody fusion (L2-S1). Between implantation and removal, pedicle screw torque was reduced by 58%, indicating significant loosening over time. Loosening was greater in screws with evoked EMG threshold under 11 mA, indicative of screw misplacement. A theoretical stress analysis revealed increased local stresses at the screw interface in pedicles with decreased difference in pedicle thickness and screw diameter. Loosening was greatest in vertebrae at the extremities of the fused segments, but was significantly lower in segments with one level of fusion than in those with two or more. Clinical significance These data indicate that pedicle screws can loosen significantly in patients with recurrent back pain and warrant further research into methods to reduce the incidence of screw loosening and to understand the risks and potential benefits of instrumentation removal. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2673-2681, 2017.

Neovascularization is a critical early step toward successful tissue regeneration during wound healing. While vasculature has long been recognized as highly mechanosensitive (to fluid shear, pulsatile luminal pressure, etc.), the effects of extracellular matrix strains on angiogenesis are poorly understood. Previously, we found that dynamic matrix compression in vivo potently regulated neovascular growth during tissue regeneration; however, whether matrix deformations directly regulate00 angiogenesis remained unknown. Here, we tested the effects of load initiation time, strain magnitude, and mode of compressive deformation (uniform compression vs. compressive indentation that also introduced shear stress) on neovascularization and key angiogenic and mechanotransduction signaling pathways by microvascular fragments in vitro. We hypothesized that neovascularization would be enhanced by delayed, moderate compression and inhibited by early, high magnitude compression and by compressive indentation. Consistent with our hypothesis, early, high magnitude loading inhibited vessel growth, while delayed loading enhanced vessel growth. Compressive indentation led to longer, more branched networks than uniform compression – particularly at high strain magnitude. Gene expression was differentially regulated by time of load initiation; genes associated with active angiogenic sprouts were downregulated by early loading but upregulated by delayed loading. Canonical gene targets of the YAP/TAZ mechanotransduction pathway were increased by loading and abrogated by pharmacological YAP inhibition. Together, these data demonstrate that neovascularization is directly responsive to dynamic matrix strain and is particularly sensitive to the timing of load initiation. This work further identifies putative mechanoregulatory angiogenic mechanisms and implicates a critical role for dynamic mechanical cues in vascularized tissue regeneration.Statement of SignificanceMechanical cues influence tissue regeneration, and although vasculature is known to be mechanically sensitive, remarkably little is known about the effects of bulk extracellular matrix deformation on the nascent vessel networks found in healing tissues. Here, we demonstrated that load initiation time, magnitude, and mode all regulate microvascular growth, as well as upstream angiogenic and mechanotransduction signaling pathways. Across all tested magnitudes and modes, microvascular network formation and upstream signaling were powerfully regulated by the timing of load initiation. This work provides a new foundational understanding of how extracellular matrix mechanics regulate angiogenesis and has critical implications for clinical translation of new regenerative medicine therapies and physical rehabilitation strategies designed to enhance revascularization during tissue regeneration.

Mesenchymal condensations promote defect repair by mimicking cellular, morphogenetic, and mechanical aspects of bone development., Endochondral ossification during long bone development and natural fracture healing initiates by mesenchymal cell condensation, directed by local morphogen signals and mechanical cues. Here, we aimed to mimic development for regeneration of large bone defects. We hypothesized that engineered human mesenchymal condensations presenting transforming growth factor–β1 (TGF-β1) and/or bone morphogenetic protein-2 (BMP-2) from encapsulated microparticles promotes endochondral defect regeneration contingent on in vivo mechanical cues. Mesenchymal condensations induced bone formation dependent on morphogen presentation, with BMP-2 + TGF-β1 fully restoring mechanical function. Delayed in vivo ambulatory loading significantly enhanced the bone formation rate in the dual morphogen group. In vitro, BMP-2 or BMP-2 + TGF-β1 initiated robust endochondral lineage commitment. In vivo, however, extensive cartilage formation was evident predominantly in the BMP-2 + TGF-β1 group, enhanced by mechanical loading. Together, this study demonstrates a biomimetic template for recapitulating developmental morphogenic and mechanical cues in vivo for tissue engineering.

Large bone defects cannot form a callus and exhibit high complication rates even with the best treatment strategies available. Tissue engineering approaches often use scaffolds designed to match the properties of mature bone. However, natural fracture healing is most efficient when it recapitulates development, forming bone via a cartilage intermediate (endochondral ossification). Because mechanical forces are critical for proper endochondral bone development and fracture repair, we hypothesized that recapitulating developmental mechanical forces would be essential for large bone defect regeneration in rats. Here, we engineered mesenchymal condensations that mimic the cellular organization and lineage progression of the early limb bud in response to local transforming growth factor-β1 presentation from incorporated gelatin microspheres. We then controlled mechanical loading in vivo by dynamically tuning fixator compliance. Mechanical loading enhanced mesenchymal condensation-induced endochondral bone formation in vivo, restoring functional bone properties when load initiation was delayed to week 4 after defect formation. Live cell transplantation produced zonal human cartilage and primary spongiosa mimetic of the native growth plate, whereas condensation devitalization before transplantation abrogated bone formation. Mechanical loading induced regeneration comparable to high-dose bone morphogenetic protein-2 delivery, but without heterotopic bone formation and with order-of-magnitude greater mechanosensitivity. In vitro, mechanical loading promoted chondrogenesis and up-regulated pericellular matrix deposition and angiogenic gene expression. In vivo, mechanical loading regulated cartilage formation and neovascular invasion, dependent on load timing. This study establishes mechanical cues as key regulators of endochondral bone defect regeneration and provides a paradigm for recapitulating developmental programs for tissue engineering.

Bone fragility fractures are caused by low bone mass or impaired bone quality. Osteoblast/osteoclast coordination determines bone mass, but the factors that control bone quality are poorly understood. Osteocytes regulate osteoblast and osteoclast activity on bone surfaces but can also directly reorganize the bone matrix to improve bone quality through perilacunar/canalicular remodeling; however, the molecular mechanisms remain unclear. We previously found that deleting the transcriptional regulators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-motif (TAZ) from osteoblast-lineage cells caused lethality in mice due to skeletal fragility. Here, we tested the hypothesis that YAP and TAZ regulate osteocyte-mediated bone remodeling by conditional ablation of both YAP and TAZ from mouse osteocytes using 8 kb-DMP1-Cre. Osteocyte-conditional YAP/TAZ deletion reduced bone mass and dysregulated matrix collagen content and organization, which together decreased bone mechanical properties. Further, YAP/TAZ deletion impaired osteocyte perilacunar/canalicular remodeling by reducing canalicular network density, length, and branching, as well as perilacunar flourochrome-labeled mineral deposition. Consistent with recent studies identifying TGF-β as a key inducer of osteocyte expression of matrix-remodeling enzymes, YAP/TAZ deletion in vivo decreased osteocyte expression of matrix proteases MMP13, MMP14, and CTSK. In vitro, pharmacologic inhibition of YAP/TAZ transcriptional activity in osteocyte-like cells abrogated TGF-β-induced matrix protease gene expression. Together, these data show that YAP and TAZ control bone matrix accrual, organization, and mechanical properties by regulating osteocyte-mediated bone remodeling. Elucidating the signaling pathways that control perilacunar/canalicular remodeling may enable future therapeutic targeting of bone quality to reverse skeletal fragility. © 2019 American Society for Bone and Mineral Research.

Osteocytes are bone matrix-entombed cells that form an interconnected network of processes called the lacunar/canalicular system, which enables osteocytes to coordinate bone formation and resorption. Osteocytes indirectly regulate osteoblast and osteoclast activity on bone surfaces but also directly resorb and deposit their surrounding bone matrix through perilacunar/canalicular remodeling. However, the molecular mechanisms by which osteocytes control bone remodeling remain unclear. We previously reported that the transcriptional regulators Yes-associated protein (YAP) and Transcriptional co-activator with PDZ-motif (TAZ) promote bone acquisition in osteoblast-lineage cells. Here, we tested the hypothesis that YAP and TAZ regulate osteocyte-mediated bone remodeling by conditional ablation of both YAP and TAZ from mouse osteocytes using 8kb-DMP1-Cre. Osteocyte conditional YAP/TAZ deletion reduced bone mass and dysregulated matrix collagen content and organization, which together impaired bone mechanical properties. YAP/TAZ deletion reduced osteoblast number and activity and increased osteoclast activity. In addition, YAP/TAZ deletion directly impaired osteocyte lacunar/canalicular network remodeling, reducing canalicular density, length, and branching, but did not alter lacunar size or shape. Further, consistent with recent studies identifying TGF-β signaling as a key inducer of perilacunar/canalicular remodeling through expression of matrix-remodeling enzymes, YAP/TAZ deletion in vivo decreased osteocyte expression of matrix proteases Mmp13, Mmp14, and Cathepsin K. In vitro, pharmacologic inhibition of YAP/TAZ transcriptional activity in osteocyte-like cells abrogated TGF-β-induced protease gene expression. Together, these data show that YAP and TAZ act downstream of TGF-β in osteocytes to control bone matrix accrual, organization, and mechanical properties indirectly by coordinating osteoblast/osteoclast activity and directly by regulating perilacunar/canalicular remodeling.

Endochondral ossification during long bone development and natural fracture healing initiates by mesenchymal cell condensation and is directed by local morphogen signals and mechanical cues. Here, we aimed to mimic these developmental conditions for regeneration of large bone defects. We hypothesized that engineered human mesenchymal stem cell (hMSC) condensations with in situ presentation of transforming growth factor-β1 (TGF-β1) and/or bone morphogenetic protein-2 (BMP-2) from encapsulated microparticles would promote endochondral regeneration of critical-sized rat femoral bone defects in a manner dependent on the in vivo mechanical environment. Mesenchymal condensations induced bone formation dependent on morphogen presentation, with dual BMP-2 + TGF-β1 fully restoring mechanical bone function by week 12. In vivo ambulatory mechanical loading, initiated at week 4 by delayed unlocking of compliant fixation plates, significantly enhanced the bone formation rate in the four weeks after load initiation in the dual morphogen group. In vitro, local presentation of either BMP-2 alone or BMP-2 + TGF-β1 initiated endochondral lineage commitment of mesenchymal condensations, inducing both chondrogenic and osteogenic gene expression through SMAD3 and SMAD5 signaling. In vivo, however, endochondral cartilage formation was evident only in the BMP-2 + TGF-β1 group and was enhanced by mechanical loading. The degree of bone formation was comparable to BMP-2 soaked on collagen but without the ectopic bone formation that limits the clinical efficacy of BMP-2/collagen. In contrast, mechanical loading had no effect on autograft-mediated repair. Together, this study demonstrates a biomimetic template for recapitulating developmental morphogenic and mechanical cues in vivo for tissue engineering.One Sentence SummaryMimicking aspects of the cellular, biochemical, and mechanical environment during early limb development, chondrogenically-primed human mesenchymal stem cell condensations promoted functional healing of critical-sized femoral defects via endochondral ossification, and healing rate and extent was a function of the in vivo mechanical environment.

Optogenetic tools are created to control RhoA GTPase, a central regulator of actin organization and actomyosin contractility. RhoA GTPase, or its upstream activator ARHGEF11, is fused to BcLOV4, a photoreceptor that can be dynamically recruited to the plasma membrane by a light-regulated protein-lipid electrostatic interaction with the inner leaflet. Direct membrane recruitment of these proteins induces potent contractile signaling sufficient to separate adherens junctions with as little as one pulse of blue light. Induced cytoskeletal morphology changes are dependent on the alignment of the spatially patterned stimulation with the underlying cell polarization. RhoA-mediated cytoskeletal activation drives yes-associated protein (YAP) nuclear localization within minutes and consequent mechanotransduction verified by YAP-transcriptional enhanced associate domain transcriptional activity. These single-transgene tools do not require protein binding partners for dynamic membrane localization and permit spatiotemporally precise control over RhoA signaling to advance the study of its diverse regulatory roles in cell migration, morphogenesis, and cell cycle maintenance.

Insufficient blood vessel supply is a primary limiting factor for regenerative approaches to large bone defect repair. Recombinant BMP-2 delivery induces robust bone formation and has been observed to enhance neovascularization, but whether the angiogenic effects of BMP-2 are due to direct endothelial cell stimulation or to indirect paracrine signaling remains unclear. Here, we evaluated the effects of BMP-2 delivery on vascularized bone regeneration and tested whether BMP-2 induces neovascularization directly or indirectly. We found that delivery of BMP-2 (5 μg) enhanced both bone formation and neovascularization in critically sized (8 mm) rat femoral bone defects; however, BMP-2 did not directly stimulate angiogenesisin vitro. In contrast, conditioned medium from both mesenchymal progenitor cells and osteoblasts induced angiogenesisin vitro, suggesting a paracrine mechanism of BMP-2 action. Consistent with this inference, co-delivery of BMP-2 with endothelial colony forming cells (ECFCs) to a heterotopic site, distant from the bone marrow niche, induced ossification but had no effect on neovascularization. Taken together, these data suggest that BMP-2 induces neovascularization during bone regeneration primarily through paracrine activation of osteoprogenitor cells.

Atrophic nonunion represents an extremely challenging clinical dilemma for both physicians and fracture patients alike, but its underlying mechanisms are still largely unknown. Here, we established a mouse model that recapitulates clinical atrophic nonunion through the administration of focal radiation to the long bone midshaft 2 weeks before a closed, semistabilized, transverse fracture. Strikingly, fractures in previously irradiated bone showed no bony bridging with a 100% nonunion rate. Radiation triggered distinct repair responses, separated by the fracture line: a less robust callus formation at the proximal side (close to the knee) and bony atrophy at the distal side (close to the ankle) characterized by sustained fibrotic cells and type I collagen-rich matrix. These fibrotic cells, similar to human nonunion samples, lacked osteogenic and chondrogenic differentiation and exhibited impaired blood vessel infiltration. Mechanistically, focal radiation reduced the numbers of periosteal mesenchymal progenitors and blood vessels and blunted injury-induced proliferation of mesenchymal progenitors shortly after fracture, with greater damage particularly at the distal side. In culture, radiation drastically suppressed proliferation of periosteal mesenchymal progenitors. Radiation did not affect hypoxia-induced periosteal cell chondrogenesis but greatly reduced osteogenic differentiation. Lineage tracing using multiple reporter mouse models revealed that mesenchymal progenitors within the bone marrow or along the periosteal bone surface did not contribute to nonunion fibrosis. Therefore, we conclude that atrophic nonunion fractures are caused by severe damage to the periosteal mesenchymal progenitors and are accompanied by an extraskeletal, fibro-cellular response. In addition, we present this radiation-induced periosteal damage model as a new, clinically relevant tool to study the biologic basis of therapies for atrophic nonunion. © 2018 American Society for Bone and Mineral Research.

Cell migration initiates by traction generation through reciprocal actomyosin tension and focal adhesion reinforcement, but continued motility requires adaptive cytoskeletal remodeling and adhesion release. Here, we asked whetherde novogene expression contributes to this cytoskeletal feedback. We found that global inhibition of transcription or translation does not impair initial cell polarization or migration initiation, but causes eventual migratory arrest through excessive cytoskeletal tension and over-maturation of focal adhesions, tethering cells to their matrix. The transcriptional co-activators YAP and TAZ mediate this feedback response, modulating cell mechanics by limiting cytoskeletal and focal adhesion maturation to enable persistent cell motility and 3D vasculogenesis. Motile arrest after YAP/TAZ ablation was partially rescued by depletion of the YAP/TAZ-dependent myosin phosphatase regulator, NUAK2, or by inhibition of Rho-ROCK-myosin II. Together, these data establish a transcriptional feedback axis necessary to maintain a responsive cytoskeletal equilibrium and persistent migration.

The functions of the paralogous transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in bone are controversial. Each has been observed to promote or inhibit osteogenesis in vitro, with reports of both equivalent and divergent functions. Their combinatorial roles in bone physiology are unknown. We report that combinatorial YAP/TAZ deletion from skeletal lineage cells, using Osterix-Cre, caused an osteogenesis imperfecta-like phenotype with severity dependent on allele dose and greater phenotypic expressivity with homozygous TAZ vs. YAP ablation. YAP/TAZ deletion decreased bone accrual and reduced intrinsic bone material properties through impaired collagen content and organization. These structural and material defects produced spontaneous fractures, particularly in mice with homozygous TAZ deletion and caused neonatal lethality in dual homozygous knockouts. At the cellular level in vivo, YAP/TAZ ablation reduced osteoblast activity and increased osteoclast activity, in an allele dose-dependent manner, impairing bone accrual and remodeling. Transcriptionally, YAP/TAZ deletion and small-molecule inhibition of YAP/TAZ interaction with the transcriptional coeffector TEAD reduced osteogenic and collagen-related gene expression, both in vivo and in vitro. These data demonstrate that YAP and TAZ combinatorially promote bone development through regulation of osteoblast activity, matrix quality, and osteoclastic remodeling.-Kegelman, C. D., Mason, D. E., Dawahare, J. H., Horan, D. J., Vigil, G. D., Howard, S. S., Robling, A. G., Bellido, T. M., Boerckel, J. D. Skeletal cell YAP and TAZ combinatorially promote bone development.

The regeneration of bone in segmental defects has historically been a challenge in the orthopedic field. In particular, a lack of vascular supply often leads to nonunion and avascular necrosis. While the gold standard of clinical care remains the autograft, this approach is limited for large bone defects. Therefore, allograft bone is often required for defects of critical size though a high complication rate is directly attributable to their limited ability to revitalize, revascularize, and remodel resulting in necrosis and re-fracture. However, emerging insights into the mechanisms of bone healing continue to expand treatment options for bony defects to include synthetic materials, growth factors, and cells. The success of such strategies hinges on fabricating an environment that can mimic the body’s natural healing process, allowing for vascularization, bridging, and remodeling of bone. Biological, chemical, and engineering techniques have been explored to determine the appropriate materials and factors for potential use. This review will serve to highlight some of the historical and present uses of allografts and autografts and current strategies in bone tissue engineering for the treatment for bony defects, with particular emphasis on vascularization.

Bone is a dynamic tissue that can adapt its architecture in response to mechanical signals under the control of osteocytes, which sense mechanical deformation of the mineralized bone. However, cells in the marrow are also mechanosensitive and may contribute to load-induced bone adaptation, as marrow is subjected to mechanical stress during bone deformation. We investigated the contribution of mechanotransduction in marrow cells to trabecular bone formation by applying low magnitude mechanical stimulation (LMMS) to porcine vertebral trabecular bone explants in an in situ bioreactor. The bone formation rate was higher in stimulated explants compared to unloaded controls which represent a disuse condition (CNT). However, sclerostin protein expression in osteocytes was not different between groups, nor was expression of osteocytic mechanoregulatory genes SOST, IGF-1, CTGF, and Cyr61, suggesting the mechanoregulatory program of osteocytes was unaffected by the loading regime. In contrast, c-Fos, a gene indicative of mechanical stimulation, was upregulated in the marrow cells of mechanically stimulated explants, while the level of activated c-Jun decreased by 25%. The activator protein 1 (AP-1) transcription factor is a heterodimer of c-Fos and c-Jun, which led us to investigate the expression of the downstream target gene cyclin-D1, a gene associated with cell cycle progression and osteogenesis. Cyclin-D1 gene expression in the stimulated marrow was approximately double that of the controls. The level of phosphorylated PYK2, a purported inhibitor of osteoblast differentiation, also decreased in marrow cells from stimulated explants. Taken together, mechanotransduction in marrow cells induced trabecular bone formation independent of osteocyte signaling. Identifying the specific cells and signaling pathways involved, and verifying them with inhibition of specific signaling molecules, could lead to potential therapeutic targets for diseases characterized by bone loss.

Large bone defects cannot heal without intervention and have high complication rates even with the best treatments available. In contrast, bone fractures naturally healing with high success rates by recapitulating the process of bone development through endochondral ossification.1 Endochondral tissue engineering may represent a promising paradigm, but large bone defects are unable to naturally form a callus. We engineered mesenchymal condensations featuring local morphogen presentation (TGF-β1) to mimic the cellular organization and lineage progression of the early limb bud. As mechanical forces are 2,3 critical for proper endochondral ossification during bone morphogenesis2,3 and fracture healing, we hypothesized that mechanical cues would be important for endochondral regeneration.4,5 Here, using fixation plates that modulate ambulatory load transfer through dynamic tuning of axial compliance, we found that in vivo mechanical loading was necessary to restore bone function to large bone defects through endochondral ossification. Endochondral regeneration produced zonal cartilage and primary spongiosa mimetic of the native growth plate. Live human chondrocytes contributed to endochondral regeneration in vivo, while cell devitalization prior to condensation transplantation abrogated bone formation. Mechanical loading induced regeneration comparable to high-dose BMP-2 delivery, but without heterotopic bone formation and with order-of-magnitude greater mechanosensitivity.6–8In vitro, mechanical loading promoted chondrogenesis, and upregulated pericellular collagen 6 deposition and angiogenic gene expression. Consistently, in vivo mechanical loading regulated cartilage formation and neovascular invasion dependent on load timing. Together, this study represents the first demonstration of the effects of mechanical loading on transplanted cell-mediated bone defect regeneration, and provides a new template for recapitulating developmental programs for tissue engineering.

The functions of the transcriptional co-activators YAP and TAZ in bone are controversial. Each has been observed to either promote or inhibit osteogenesisin vitro, while their roles in bone development are unknown. Here we report that combinatorial YAP/TAZ deletion from skeletal cells in mice caused osteogenesis imperfecta with severity dependent on targeted cell lineage and allele dosage. Osteocyte-conditional deletion impaired bone accrual and matrix collagen, while allele dosage-dependent deletion from all osteogenic lineage cells caused spontaneous fractures, with neonatal lethality only in dual homozygous knockouts. We identified putative target genes whose mutation in humans causes osteogenesis imperfecta and which contain promoter-proximate binding domains for the YAP/TAZ co-effector, TEAD4. Two candidates, Col1a1 and SerpinH1, exhibited reduced expression upon either YAP/TAZ deletion or YAP/TAZ-TEAD inhibition by verteporfin. Together, these data demonstrate that YAP and TAZ redundantly promote bone matrix development and implicate YAP/TAZ-mediated transcriptional regulation of collagen in osteogenesis imperfecta.

The fields of developmental biology and tissue engineering have been revolutionized in recent years by technological advancements, expanded understanding, and biomaterials design, leading to the emerging paradigm of "developmental" or "biomimetic" tissue engineering. While developmental biology and tissue engineering have long overlapping histories, the fields have largely diverged in recent years at the same time that crosstalk opportunities for mutual benefit are more salient than ever. In this perspective article, we will use musculoskeletal development and tissue engineering as a platform on which to discuss these emerging crosstalk opportunities and will present our opinions on the bright future of these overlapping spheres of influence. The multicellular programs that control musculoskeletal development are rapidly becoming clarified, represented by shifting paradigms in our understanding of cellular function, identity, and lineage specification during development. Simultaneously, advancements in bioartificial matrices that replicate the biochemical, microstructural, and mechanical properties of developing tissues present new tools and approaches for recapitulating development in tissue engineering. Here, we introduce concepts and experimental approaches in musculoskeletal developmental biology and biomaterials design and discuss applications in tissue engineering as well as opportunities for tissue engineering approaches to inform our understanding of fundamental biology. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2356-2368, 2017.

Objective— Angiogenesis is the formation of new blood vessels through endothelial cell sprouting. This process requires the mitogen-activated protein kinases, signaling molecules that are negatively regulated by the mitogen-activated protein kinase phosphatase-1 (MKP-1). The purpose of this study was to evaluate the role of MKP-1 in neovascularization in vivo and identify associated mechanisms in endothelial cells. Approach and Results— We used murine hindlimb ischemia as a model system to evaluate the role of MKP-1 in angiogenic growth, remodeling, and arteriogenesis in vivo. Genomic deletion of MKP-1 blunted angiogenesis in the distal hindlimb and microvascular arteriogenesis in the proximal hindlimb. In vitro, endothelial MKP-1 depletion/deletion abrogated vascular endothelial growth factor–induced migration and tube formation, and reduced proliferation. These observations establish MKP-1 as a positive mediator of angiogenesis and contrast with the canonical function of MKP-1 as a mitogen-activated protein kinase phosphatase, implying an alternative mechanism for MKP-1–mediated angiogenesis. Cloning and sequencing of MKP-1–bound chromatin identified localization of MKP-1 to exonic DNA of the angiogenic chemokine fractalkine, and MKP-1 depletion reduced histone H3 serine 10 dephosphorylation on this DNA locus and blocked fractalkine expression. In vivo, MKP-1 deletion abrogated ischemia-induced fractalkine expression and macrophage and T-lymphocyte infiltration in distal hindlimbs, whereas fractalkine delivery to ischemic hindlimbs rescued the effect of MKP-1 deletion on neovascular hindlimb recovery. Conclusions— MKP-1 promoted angiogenic and arteriogenic neovascular growth, potentially through dephosphorylation of histone H3 serine 10 on coding-region DNA to control transcription of angiogenic genes, such as fractalkine. These observations reveal a novel function for MKP-1 and identify MKP-1 as a potential therapeutic target.

article Although severe extremity trauma is often inclusive of skeletal and vascular damage in combination, segmen- tal bone defect repair with concomitant vascular injury has yet to be experimentally investigated. To this end, we developed a novel rat composite limb injury model by combining a critically-sized segmental bone defect with surgically-induced hind limb ischemia (HLI). Unilateral 8 mm femoral defects were created alone (BD) or in combination with HLI (BD + HLI), and all defects were treated with rhBMP-2 via a hybrid biomaterial delivery system. Based on reported clinical and experimental observations on the importance of vascular net- works in bone repair, we hypothesized that HLI would impair bone regeneration. Interestingly, the BD + HLI group displayed improved radiographic bridging, and quantitative micro-CT analysis revealed enhanced bone regeneration as early as week 4 (p b 0.01) that was sustained through week 12 (p b 0.001) and confirmed histologically. This effect was observed in two independent studies and at two different doses of rhBMP-2. Micro-CT angiography was used to quantitatively evaluate vascular networks at week 12 in both the thigh and the regenerated bone defect. No differences were found between groups in total blood vessel volume in the thigh, but clear differences in morphology were present as the BD + HLI group possessed a more interconnected network of smaller diameter vessels (p b 0.001). Accordingly, while the overall thigh vessel volume was comparable between groups, the contributions to vessel volume based on vessel diameter dif- fered significantly. Despite this evidence of a robust neovascular response in the thigh of the BD + HLI group, differences were not observed between groups for bone defect blood vessel volume or morphology. In total, our results demonstrate that a transient ischemic insult and the subsequent recovery response to HLI significantly enhanced BMP-2-mediated segmental bone defect repair, providing additional complexity to the relationship between vascular tissue networks and bone healing. Ultimately, a better understanding of the coupling mechanisms may reveal important new strategies for promoting bone healing in challenging clinical scenarios.