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

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.

ABSTRACTLarge 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.1Endochondral 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 morphogenesis2,3and fracture healing, we hypothesized that mechanical cues would be important for endochondral regeneration.4,5Here, using fixation plates that modulate ambulatory load transfer through dynamic tuning of axial compliance, we found thatin vivomechanical 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 regenerationin 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 vivomechanical 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.

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 angiogenesis in vitro . In contrast, conditioned medium from both mesenchymal progenitor cells and osteoblasts induced angiogenesis in 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.

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 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.

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.

Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the super-resolution microscopy is not feasible in many applications. In this paper, we propose and demonstrate a saturation-based super-resolution fluorescence microscopy technique that can be easily implemented and requires neither additional hardware nor complex post-processing. The method is based on the principle of stepwise optical saturation (SOS), where M steps of raw fluorescence images are linearly combined to generate an image with a [Formula: see text]-fold increase in resolution compared with conventional diffraction-limited images. For example, linearly combining (scaling and subtracting) two images obtained at regular powers extends the resolution by a factor of 1.4 beyond the diffraction limit. The resolution improvement in SOS microscopy is theoretically infinite but practically is limited by the signal-to-noise ratio. We perform simulations and experimentally demonstrate super-resolution microscopy with both one-photon (confocal) and multiphoton excitation fluorescence. We show that with the multiphoton modality, the SOS microscopy can provide super-resolution imaging deep in scattering samples.

The functions of the transcriptional co-activators YAP and TAZ in bone are controversial. Each has been observed to either promote or inhibit osteogenesis in 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.

Biologics can improve bone formation, but may diffuse away from sites of therapeutic need. We developed a click-chemistry hydrogel that rapidly polymerizes in situ to control delivery of biologics during post-suturectomy resynostosis in 21-day-old male mice. Here, we used this model to determine the role of angiogenesis in post-suturectomy resynostosis and examine whether controlled release of angiogenesis inhibitors could delay bone regeneration. Hydrogels [DB-co-PEG/poly (TEGDMA)-co-(N3-TEGDMA)] were produced containing anti-angiogenic compounds [anti-VEGFA-antibody or hypoxia inducible factor 1α-inhibitor topotecan]. Bioactivity in vitro was assessed by tube length and branching points of endothelial cells in hydrogel-conditioned media. In vivo effects were examined 14 day post-suturectomy, based on the temporal analysis of angiogenic mRNAs during resynostosis following posterior frontal suture removal. MicroCT was used to quantify angiogenesis in contrast-agent-perfused blood vessels and bone defect size in defects receiving hydrogel, anti-VEGFA/hydrogel, or topotecan/hydrogel. Shorter endothelial tube length and less branching were seen in inhibitor-conditioned media (topotecan > AbVEGFA). In vivo, both compounds inhibited angiogenesis compared with hydrogel-only. Anti-VEGFA/hydrogel reduced resynostosis compared with empty defects, but topotecan/hydrogel blocked bone regeneration. We demonstrate that anti-angiogenic compounds can be incorporated into a spontaneously polymerizing hydrogel and remain active over 14 days in vitro and in vivo. Moreover, bone formation can be delayed by inhibiting neovascularization, suggesting possible use as a therapeutic to control resynostosis following suturectomies and potential applications in other conditions where rapid osteogenesis is not desired. © 2017 Wiley Periodicals Inc. J Biomed Mater Res Part A: 105A: 2742-2749, 2017.

Bone is one of the most common and most dangerous sites for metastatic growth across cancer types, and bone metastasis remains incurable. Unfortunately, the processes by which cancers preferentially metastasize to bone are still not well understood. In this review, we summarize the morphological features, physical properties, and cell signaling events that make bone a unique site for metastasis and bone remodeling. The signaling crosstalk between the tumor cells and bone cells begins a vicious cycle - a self-sustaining feedback loop between the tumor cells and the bone microenvironment composed of osteoclasts, osteoblasts, other bone marrow cells, bone matrix, and vasculature to support both tumor growth and bone destruction. Through this crosstalk, bone provides a fertile microenvironment that can harbor dormant tumor cells, sometimes for long periods, and support their growth by releasing cytokines as the bone matrix is destroyed, similar to providing nutrients for a seed to germinate in soil. However, few models exist to study the late stages of bone colonization by metastatic tumor cells. We describe some of the current methodologies used to study bone metastasis, highlighting the limitations of these methods and alternative future strategies to be used to study bone metastasis. While in vivo animal and patient studies may provide the gold standard for studying metastasis, ex vivo models can be used as an alternative to enable more controlled experiments designed to study the late stages of bone metastasis.

Physical cues such as the mechanical properties of the extracellular matrix are known to modulate blood vessel formation, but the underlying mechanisms remain poorly understood. Yes-associated protein (YAP) and transcriptional activator with PDZ-binding domain (TAZ) were recently identified as key mediators of cellular mechanotransduction [1], but the roles of YAP and TAZ in neovascular mechanobiology have not been investigated. We hypothesized that YAP and TAZ mediate neovascular mechanotransduction of extracellular matrix rigidity by vasculogenic endothelial colony forming cells (ECFCs) cultured on/in matrices of controllable rigidity. YAP and TAZ depletion abrogated ECFC migration (Fig. 1A), and YAP/TAZ expression and nuclear localization (i.e., activation) was significantly upregulated in migrating cells (Fig. 1B). Combinatorial depletion also abrogated tubular network formation and 3D vasculogenesis in collagen matrices of variable rigidity but constant matrix density (data not shown). In 2D, PDMS rigidity regulated YAP/TAZ subcellular localization (data not shown). In 3D, vasculogenesis was enhanced in stiff (G’= 1049 Pa) collagen oligomer matrices compared to soft (G’ = 305 Pa) monomer matrices formulated at constant collagen matrix density. YAP and TAZ were nuclear localized (activated) in the stiff oligomer matrices, but cytosolic in soft monomer matrices (Fig. 1C). Mechanistically, YAP and TAZ depletion reduced cytoskeletal stress fiber formation (data not shown), implicating YAP and TAZ in a feedback mechanism to control cytoskeletal remodeling. Delivered in oligomer matrices in vivo , ECFCs formed a functional human neovascular plexus and exhibited strong expression and nuclear localization of YAP and TAZ and expression of downstream genes, Cyr61 and CTGF (data not shown). In summary, YAP and TAZ are essential for mechanical control of ECFC migration and vasculogenesis. [1] Dupont et al. 2011 Nature. 474:179-185.

A common design constraint in functional tissue engineering is that scaffolds intended for use in load-bearing sites possess similar mechanical properties to the replaced tissue. Here, we tested the hypothesis that in vivo loading would enhance bone morphogenetic protein-2 (BMP-2)-mediated bone regeneration in the presence of a load-bearing PLDL scaffold, whose pores and central core were filled with BMP-2-releasing alginate hydrogel. First, we evaluated the effects of in vivo mechanical loading on bone regeneration in the structural scaffolds. Second, we compared scaffold-mediated bone regeneration, independent of mechanical loading, with alginate hydrogel constructs, without the structural scaffold, that have been shown previously to facilitate in vivo mechanical stimulation of bone formation. Contrary to our hypothesis, mechanical loading had no effect on bone formation, distribution, or biomechanical properties in structural scaffolds. Independent of loading, the structural scaffolds reduced bone formation compared to non-structural alginate, particularly in regions in which the scaffold was concentrated, resulting in impaired functional regeneration. This is attributable to a combination of stress shielding by the scaffold and inhibition of cellular infiltration and tissue ingrowth. Collectively, these data question the necessity of scaffold similarity to mature tissue at the time of implantation and emphasize development of an environment conducive to cellular activation of matrix production and ultimate functional regeneration.

Microcomputed tomography (microCT) has become a standard and essential tool for quantifying structure-function relationships, disease progression, and regeneration in preclinical models and has facilitated numerous scientific and bioengineering advancements over the past 30 years. In this article, we recount the early events that led to the initial development of microCT and review microCT approaches for quantitative evaluation of bone, cartilage, and cardiovascular structures, with applications in fundamental structure-function analysis, disease, tissue engineering, and numerical modeling. Finally, we address several next-generation approaches under active investigation to improve spatial resolution, acquisition time, tissue contrast, radiation dose, and functional and molecular information.