The year was c.700 BC. King Hezekiah scrambled to prepare his city for a siege. But there was a problem. The Gihon spring that provided water to the hill‐top city was outside the city walls, so ...Hezekiah devised a last‐ditch plan: dig a tunnel to direct the water into the city. With time slipping away, workers dug from both ends, hoping to meet in the middle. Despite a few changes of direction, the engineers were successful; the pool filled with water, and the city successfully held off the siege. This blind tunneling through rock, from both ends, meeting in the middle, occurs more than a trillion times in each human skeleton in the osteocyte lacunar‐canalicular network. But how do osteocytes accomplish this feat? In this talk, we’ll explore the mechanisms by which osteocytes dig these tunnels, and what this might mean for bone health and disease.
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.
•Osteocytes are the mechanosensory cells in bone, and their mechanotransductive functions follow their morphological form.•Here we summarize the mechanisms that govern osteocyte differentiation, morphogenesis, mechanotransduction, and matrix remodeling.•We postulate that perilacunar/canalicular remodeling in osteocytes represents the reactivation of the osteocytes developmental program.
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 treatment of challenging fractures and large osseous defects presents a formidable problem for orthopaedic surgeons. Tissue engineering/regenerative medicine approaches seek to solve this problem ...by delivering osteogenic signals within scaffolding biomaterials. In this study, we introduce a hybrid growth factor delivery system that consists of an electrospun nanofiber mesh tube for guiding bone regeneration combined with peptide-modified alginate hydrogel injected inside the tube for sustained growth factor release. We tested the ability of this system to deliver recombinant bone morphogenetic protein-2 (rhBMP-2) for the repair of critically-sized segmental bone defects in a rat model. Longitudinal μ-CT analysis and torsional testing provided quantitative assessment of bone regeneration. Our results indicate that the hybrid delivery system resulted in consistent bony bridging of the challenging bone defects. However, in the absence of rhBMP-2, the use of nanofiber mesh tube and alginate did not result in substantial bone formation. Perforations in the nanofiber mesh accelerated the rhBMP-2 mediated bone repair, and resulted in functional restoration of the regenerated bone. μ-CT based angiography indicated that perforations did not significantly affect the revascularization of defects, suggesting that some other interaction with the tissue surrounding the defect such as improved infiltration of osteoprogenitor cells contributed to the observed differences in repair. Overall, our results indicate that the hybrid alginate/nanofiber mesh system is a promising growth factor delivery strategy for the repair of challenging bone injuries.
New vascular network formation is a critical step in the wound healing process and a primary limiting factor in functional tissue regeneration. Like many tissues, neovascular networks have been shown ...in vitro to be highly sensitive to mechanical conditions; however, the effects of matrix deformations on neovascular network formation and remodeling in engineered tissue regeneration in vivo have not been evaluated. We quantified the effects of early and delayed functional loading on neovascular growth in a rat model of large bone defect regeneration using compliant fixation plates that were unlocked to allow transfer of ambulatory loads to the defect either at the time of implantation (early), or after 4 wk of stiff fixation (delayed). Neovascular growth and bone regeneration were quantitatively evaluated 3 wk after the onset of loading by contrast-enhanced microcomputed tomography and histology. The initial vascular response to bone injury featured robust angiogenesis and collateral vessel formation, increasing parameters such as vascular volume and connectivity while decreasing degree of anisotropy. Application of early mechanical loading significantly inhibited vascular invasion into the defect by 66% and reduced bone formation by 75% in comparison to stiff plate controls. In contrast, delaying the onset of loading by 4 wk significantly enhanced bone formation by 20% and stimulated vascular remodeling by increasing the number of large vessels and decreasing the number of small vessels. Together, these data demonstrate the mechanosensitivity of neovascular networks and highlight the capacity of biomechanical stimulation to modulate postnatal vascular growth and remodeling.
Abstract Delivery of recombinant proteins is a proven therapeutic strategy to promote endogenous repair mechanisms and tissue regeneration. Bone morphogenetic protein-2 (rhBMP-2) has been used to ...promote spinal fusion and repair of challenging bone defects; however, the current clinically-used carrier, absorbable collagen sponge, requires high doses and has been associated with adverse complications. We evaluated the hypothesis that the relationship between protein dose and regenerative efficacy depends on delivery system. First, we determined the dose-response relationship for rhBMP-2 delivered to 8-mm rat bone defects in a hybrid nanofiber mesh/alginate delivery system at six doses ranging from 0 to 5 μg. Next, we directly compared the hybrid delivery system to the collagen sponge at 0.1 and 1.0 μg. Finally, we compared the in vivo protein release properties of the two delivery methods. In the hybrid delivery system, bone volume, connectivity and mechanical properties increased in a dose-dependent manner to rhBMP-2. Consistent bridging of the defect was observed for doses of 1.0 μg and greater. Compared to collagen sponge delivery at the same 1.0 μg dose, the hybrid system yielded greater connectivity by week 4 and 2.5-fold greater bone volume by week 12. These differences may be explained by the significantly greater protein retention in the hybrid system compared to collagen sponge. This study demonstrates a clear dose-dependent effect of rhBMP-2 delivered using a hybrid nanofiber mesh/alginate delivery system. Furthermore, the effective dose was found to vary with delivery system, demonstrating the importance of biomaterial carrier properties in the delivery of recombinant proteins.
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 coeff ector 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. FASEB J. 32, 2706–2721 (2018). www.fasebj.org
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.
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