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.

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.

The water-soluble complexes of Ti(IV) with citrate are of interest in environmental, biological, and materials chemistry. The aqueous solution speciation is revealed by spectropotentiometric titration. From pH 3-8, given at least three equivalents of ligand, 3:1 citrate/titanium complexes predominate in solution with successive deprotonation of dangling carboxylates as the pH increases. In this range and under these conditions, hydroxo- or oxo-metal species are not supported by the data. At ligand/metal ratios between 1:1 and 3:1, the data are difficult to fit, and are consistent with the formation of such hydroxo- or oxo- species. Stability constants for observed species are tabulated, featuring log beta-values of 9.18 for the 1:1 complex [Ti(Hcit)](+), and 16.99, 20.41, 16.11, and 4.07 for the 3:1 complexes [Ti(H(2)cit)(3)](2-), [Ti(H(2)cit)(Hcit)(2)](4-), [Ti(Hcit)(2)(cit)](6-), and [Ti(cit)(3)](8-), respectively (citric acid = H(4)cit). Optical spectra for the species are reported. The complexes exhibit similar yet distinct spectra, featuring putative citrate-to-Ti(IV) charge-transfer absorptions (lambda(max) approximately 250-310 nm with epsilon approximately 5000-7000 M(-)(1) cm(-1)). The prevailing 3:1 citrate/titanium ratio in solution is supported by electrospray mass spectrometry data. The X-ray crystal structure of a fully deprotonated tris-citrate complex Na(8)[Ti(C(6)H(4)O(7))(3)].17H(2)O (1) (or Na(8)[Ti(cit)(3)].17H(2)O) that crystallizes from aqueous solution at pH 7-8 is reported. Compound 1 crystallizes in the triclinic space group P, with a = 11.634(2) Angstroms, b = 13.223(3) Angstroms, c = 13.291(3) Angstroms, V = 1982.9(7) Angstroms(3), and Z = 2.

Natural fracture healing recapitulates bone development through endochondral ossification, 1 resulting in clinical success rates of 90-95%. 2 However, large bone defects of critical size cannot form a callus and exhibit high rates of complication and non-union even after intervention. 3 Bone tissue engineering holds promise, but traditional approaches have focused on direct, intramembranous bone formation. 4 We propose that mimicking the endochondral process that is naturally selected for bone development and fracture repair may improve regenerative outcome.Since physical stimuli are critical for proper endochondral ossification during bone morphogenesis 5,6 and fracture healing, 7-9 mechanical loading may be essential to enable reliable endochondral defect regeneration as in callus-mediated fracture repair. Here we report that in vivo mechanical loading, via dynamically tuned fixator compliance, restored bone function through endochondral ossification of engineered human mesenchymal condensations. The condensations mimic limb bud morphogenesis in response to local morphogen presentation by incorporated gelatin microspheres. Endochondral regeneration in large defects exhibited zonal cartilage and woven bone mimetic of the native growth plate, with active YAP signaling in human . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/157362 doi: bioRxiv preprint first posted online Jun. 29, 2017; 2 hypertrophic chondrocytes in vivo. Mechanical loading regulated vascular invasion and enhanced endochondral regeneration, with an order-of-magnitude greater response to loading than that observed for intramembranous repair, 10-12 restoring intact bone properties. This study represents the first demonstration of the effects of mechanical loading on transplanted cell-mediated bone defect regeneration and establishes the importance of in vivo mechanical cues, cellular selforganization, and inductive signal presentation for recapitulation of development for tissue engineering.Long bone morphogenesis is initiated by condensation of mesenchymal cells in the early limb bud, which differentiate and mature into the cartilaginous anlage that gives rise to endochondral bone formation. This process is dependent on both local morphogen gradients and mechanical forces in utero. 6,13 Natural bone fracture healing recapitulates endochondral bone development, but only under conditions of compressive interfragmentary strain. 14,15 Without mechanical loading, fractures will heal through direct, intramembranous bone formation, 9 implicating mechanical cues as essential regulators of endochondral ossification. The emerging paradigm of biomimetic tissue engineering approaches aim to replicate this process, 16,17 but functional endochondral bone regeneration using transplanted human progenitor cells remains elusive potentially due to insuff...