| Background: It is well known that external mechanical stimulation has the capacity of increasing bone quantity and quality through regulating bone remodeling,whereas the loss of physiological machinery(e.g.,microgravity,bed rest and plaster fixation)induces rapid loss in both bone quantity and quality.Bone tissues experience many different types of physiological mechanical stimulation,such as compressive forces,fluid shear stress and mechanical stretch.Mechanical stretch is an important mechanical loading pattern in clinical application,such as distraction osteogenesis for limb lengthening,orthodontic treatment,and union fracture repair and so on.Accumulating evidence suggests that mechanical stretch is able to stimulate bone anabolism and accelerate bone regeneration in vivo.Several in vitro studies have shown that mechanical stretch significantly accelerates osteoblastic maturation,differentiation,and mineralized matrix deposition,and also promotes osteogenesis-related gene expression.However,the underlying mechanism by which mechanical stretch improves osteoblastic differentiation remains poorly understood.Our previous study has demonstrated that the Akt/m TOR/p70s6 k pathway is also involved in the regulation of energy metabolism and osteogenesis differentiation in osteoblasts in response to mechanical stretch,and inhibit m TOR signal.mechanical stretch induced osteogenic differentiation was inhibited,but inhibition of Akt signal did not change significantly(except ALP),suggesting that there may be some signal interaction with m TOR signal to maintain the phenotype of osteoblasts.Moreover,both m TOR and NF-?B are the downstream molecules of Akt,and the reciprocal interconnection between m TOR and NF-?B has been identified in the regulation of tumor cell activities.Nevertheless,whether NF-?B and m TOR exhibit cross talk in osteoblast under mechanical stretch,and the role of their potential interactions in the maintenance of osteoblast homeostasis remain unknown.Methods: Firstly,based on the current mechanical stretch loading device,this research group independently developed and designed a mechanical stretch closed-loop feedback loading system which can control the deformation and frequency of mechanical stretch.Then,the relationship between pressure and deformation was analyzed by Image J software,and the effect of pressure on membrane deformation in different directions and loading cycles was studied.Then the osteoblasts were subjected to mechanical stretch(10%,6 cycles/min)with a mechanical stretch closed-loop feedback loading system.The morphology of osteoblasts was observed by ordinary optical microscope.The effects of mechanical stretch on osteogenic differentiation and mineralization were observed by q-RTPCR,ALP staining and Alizarin red staining.Finally,Western blotting and immunofluorescence techniques were used to study the effect of mechanical stretch on the protein expression of osteoblasts.Inhibitors and si RNA were used to block m TOR and NF-?B signals.q-RTPCR was used to analyze the effect of mechanical stretch on the expression of osteogenic related genes.Results: The self-designed mechanical stretch closed-loop feedback loading system has a linear relationship between pressure and deformation,the membrane deformation is isotropic,the deformation error caused by membrane compliance has no statistical significance,and the equipment can apply repeatable and sustainable mechanical stretch to cells.The results showed that osteoblasts arranged regularly and linearly under mechanical stretch.Mechanical stretch significantly increased the m RNA expression of osteogenic related genes,ALP activity and mineralized nodules.Mechanical stretch stimulation resulted in significant increase in the protein expression of p-m TOR/m TOR.After blockade of m TOR,mechanical stretch-induced increase in osteoblast differentiation-related gene expression was significantly blocked.Significant increase in the protein expression of p-p65/p65 in MG63 cells induced by mechanical stretch stimulation.Immunofluorescence results demonstrated that mechanical stretch induced nuclear translocation of p65 in MG63 cells.After blockade of NF-?B p65,mechanical stretch-induced up-regulation of osteoblast differentiation-related gene expression was further increased.The m RNA levels of osteoblast differentiation-related gene under mechanical stretch after co-inhibition of m TOR and NF-?B p65 were signficantly higher than those after single inhibition of m TOR,and significantly lower than single inhibition of p65.Similarly,the gene expression levels of osteoblast differentiation-related markers after inhibition of Akt exhibit no significant difference as compared with the co-inhibition of m TOR and p65.Conclusion: Mechanical stretch closed-loop feedback loading system is of great importance to the study of cell biomechanical signal transduction.Our findings suggested that the reciprocal interconnection between m TOR and NF-?B plays a critical role in mechanical stretch-mediated osteoblast differentiation.The results demonstrated that mechanical stretch stimulated osteoblast differentiation by both m TOR-mediated positive regulation and NF-?B-mediated negative regulation.The interaction between m TOR and NF-?B was able to maintain stretchinduced osteoblast differentiation at a moderate level(neither too higher nor too low),which might be an important signaling event in stretch-related mechanotransduction in osteoblasts.This study for the first time reveals the reciprocal interconnection between m TOR and NF-?B in osteoblasts under mechanical stretch,and indicates that the interactions between NF-?B and m TOR play a key role in the regulation of cellular homeostasis of osteoblasts in response to mechanical stretch.These findings are not only helpful for enriching our basic knowledge of the molecular mechanisms of osteoblast mechanotransduction,but also providing insight into the clinical therapeutic modality associated with mechanical stretching(such as distraction osteogenesis). |
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