Palo Alto, California, United States
Palo Alto, California, United States

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PubMed | Bone and Joint Rehabilitation nter
Type: Journal Article | Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology | Year: 2010

Primary cilia are chemosensing and mechanosensing organelles that regulate remarkably diverse processes in a variety of cells. We previously showed that primary cilia play a role in mediating mechanosensing in bone cells through an unknown mechanism that does not involve extracellular Ca(2+)-dependent intracellular Ca(2+) release, which has been implicated in all other cells that transduce mechanical signals via the cilium. Here, we identify a molecular mechanism linking primary cilia and bone cell mechanotransduction that involves adenylyl cyclase 6 (AC6) and cAMP. Intracellular cAMP was quantified in MLO-Y4 cells exposed to dynamic flow, and AC6 and primary cilia were inhibited using RNA interference. When exposed to flow, cells rapidly (<2 min) and transiently decreased cAMP production in a primary cilium-dependent manner. RT-PCR revealed differential expression of the membrane-bound isoforms of adenylyl cyclase, while immunostaining revealed one, AC6, preferentially localized to the cilium. Further studies showed that decreases in cAMP in response to flow were dependent on AC6 and Gd(3+)-sensitive channels but not intracellular Ca(2+) release and that this response mediated flow-induced COX-2 gene expression. The signaling events identified provide important details of a novel early mechanosensing mechanism in bone and advances our understanding of how signal transduction occurs at the primary cilium.


PubMed | Bone and Joint Rehabilitation nter
Type: Journal Article | Journal: Journal of biomechanics | Year: 2010

Epigenetic regulation of gene expression occurs due to alterations in chromatin proteins that do not change DNA sequence, but alter the chromatin architecture and the accessibility of genes, resulting in changes to gene expression that are preserved during cell division. Through this process genes are switched on or off in a more durable fashion than other transient mechanisms of gene regulation, such as transcription factors. Thus, epigenetics is central to cellular differentiation and stem cell linage commitment. One such mechanism is DNA methylation, which is associated with gene silencing and is involved in a cells progression towards a specific fate. Mechanical signals are a crucial regulator of stem cell behavior and important in tissue differentiation; however, there has been no demonstration of a mechanism whereby mechanics can affect gene regulation at the epigenetic level. In this study, we identified candidate DNA methylation sites in the promoter regions of three osteogenic genes from bone marrow derived mesenchymal stem cells (MSCs). We demonstrate that mechanical stimulation alters their epigenetic state by reducing DNA methylation and show an associated increase in expression. We contrast these results with biochemically induced differentiation and distinguish expression changes associated with durable epigenetic regulation from those likely to be due to transient changes in regulation. This is an important advance in stem cell mechanobiology as it is the first demonstration of a mechanism by which the mechanical micro-environment is able to induce epigenetic changes that control osteogenic cell fate, and that can be passed to daughter cells. This is a first step to understanding that will be vital to successful bone tissue engineering and regenerative medicine, where continued expression of a desired long-term phenotype is crucial.


PubMed | Washington University in St. Louis, Bone and Joint Rehabilitation nter and Stanford University
Type: Journal Article | Journal: Global spine journal | Year: 2015

Study DesignIn vitro testing. ObjectiveTo determine whether long cervical and cervicothoracic fusions increase the intradiscal pressure at the adjacent caudal disk and to determine which thoracic end vertebra causes the least increase in the adjacent-level intradiscal pressure. MethodsA bending moment was applied to six cadaveric cervicothoracic spine specimens with intact rib cages. Intradiscal pressures were recorded from C7-T1 to T9-10 before and after simulated fusion by anterior cervical plating and posterior thoracic pedicle screw constructs. The changes in the intradiscal pressure from baseline were calculated and compared. ResultsNo significant differences where found when the changes of the juxtafusion intradiscal pressure at each level were compared for the flexion, extension, and left and right bending simulations. However, combining the pressures for all directions of bending at each level demonstrated a decrease in the pressures at the T2-T3 level. Exploratory analysis comparing changes in the pressure at T2-T3 to other levels showed a significant decrease in the pressures at this level (p=0.005). ConclusionsBased on the combined intradiscal pressures alone it may be advantageous to end long constructs spanning the cervicothoracic junction at the T2 level if there are no other mitigating factors.

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