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.

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..

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...

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.

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 in mice. 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 before 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 Osx‐CretetOff) in which YAP and TAZ were deleted before 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 before 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. © 2020 American Society for Bone and Mineral Research (ASBMR).