Effect of mechano-growth factor on osteoclast activity and its mechanism / 中华创伤杂志

Objective:To investigate the effect of mechano-growth factor（MGF）on osteoclast activity and its mechanism.Methods:The RAW264.7 precursor osteoclast cell line was cultured with 25 ng/ml macrophage-colony stimulating factor（M-CSF）and 30 ng/ml receptor activator of NF-κB ligand（RANKL），and identified by tartrate resistant acid phosphatase（TRAP）staining after 7 days of culture. Western blot anslysis was used to determine the effect of 45 ng/ml MGF on the phosphoinositide-3-kinase/protein kinase B（PI3K/AKT）signaling pathway in separated osteoclasts，including levels of AKT，phosphorylation（p）-AKT，lactation mammalian target of rapamycin（mTOR），p-mTOR and TRAP at 0，4，8 and 12 hours. Real-time fluorescence quantitative PCR was used to expressions of TRAP in osteoclasts at 0，4，8 and 12 hours. The PI3K/Akt phosphorylation inhibitor LY294002（20 μmol/L）combined with MGF（45 ng/ml）was used to act on osteoclasts，and expression levels of Akt，p-Akt，mTOR，p-mTOR and TRAP were detected by Western blot at 0，4，8 and 12 hours.Results:After culturing RAW264.7 cells with M-CSF and RANKL for 7 days，a large number of osteoclasts with positive TRAP staining can be obtained. Western blot analysis showed expression levels of Akt and mTOR did not change significantly over time（ P>0.05），expression levels of p-Akt and p-mTOR increased continuously from（2.18±0.34）pg/ml and（0.83±0.10）pg/ml at 0 hour to（3.86±0.36）pg/ml and（1.56±0.19）pg/ml at 12 hours（ P<0.05），and expression level of TRAP decreased significantly over time，from（5.66±0.47）pg/ml at 0 hour to（3.76±0.38）pg/ml at 12 hours（ P<0.05）. Real-time fluorescence quantitative PCR analysis of expression of TRAP in osteoclasts showed that MGF inhibited the expression of TRAP in osteoclasts，which decreased from 1.02±0.06 at 0 hour to 0.53±0.11 at 12 hours（ P<0.05）. After acting LY294002 combined with MGF on osteoclasts，Western blot analysis showed expression levels of Akt and mTOR did not change significantly over time（ P>0.05），expression levels of p-AKT and p-mTOR decreased significantly from（3.28±0.18）pg/ml and（3.29±0.22）pg/ml at 0 hour to（2.06±0.34）pg/ml and（2.04±0.20）pg/ml at 12 hours（ P<0.05），and expression level of TRAP had no significant difference over time（ P>0.05）. Conclusions:MGF inhibits osteoclast activity by inhibiting the expression of TRAP in osteoclasts through PI3K/Akt signaling pathway. LY294002 inhibits the expression of PI3K/Akt signaling pathway in osteoclasts，further verifying the mechanism of MGF inhibiting osteoclast activity，and this finding puts forward new ideas for clinical prevention and treatment of osteoporosis.

Effect of enhanced masticatory force on OPG, RANKL and MGF in alveolar bone of ovariectomized rats

Abstract Menopause induces oral bone loss, leading to various oral diseases. Mastication importantly affects bone metabolism in the jawbone. Objective: To analyze the effect of enhanced masticatory force on osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), and mechano-growth factor (MGF) in alveolar bone of ovariectomized rats and to study the mechanics mechanism of the alveolar bone of ovariectomized rats response to enhanced masticatory force. Methodology: Thirty Sprague Dawley rats were randomly divided into three groups: sham-operation group (fat around the removed ovary + normal hard diet), model group (ovariectomy + normal hard diet), and experimental group (ovariectomy + high hard diet). It was a 2-month experiment. Enzyme-linked immunosorbent assay (ELISA) detected serum estradiol (E2), osteocalcin (BGP) and alkaline phosphatase (ALP) in rats. Bone histomorphometric indices in the third molar region of maxilla were detected by micro-CT; protein expressions of OPG, RANKL, and MGF in the third molar region of maxilla was detected by Western blot; and gene expression of OPG, RANKL, and MGF in the third molar region of maxilla was detected by Quantitative Real-Time PCR. Results: Comparing with model group, serum E2 in experimental group increased but not significantly, serum BGP and serum ALP in experimental group decreased but not significantly, OPG in experimental group in alveolar bone increased significantly, RANKL in experimental group in alveolar bone decreased significantly, RANKL/OPG ratio in experimental group decreased significantly, MGF in experimental group in alveolar bone increased significantly, bone volume to total volume fraction increased significantly in experimental group, trabecular thickness increased significantly in experimental group, and trabecular separation decreased significantly in experimental group. Conclusion: Enhanced masticatory force affected the expression of OPG, RANKL, and MGF in alveolar bone of ovariectomized rats, improved the quality of jaw bone of ovariectomized rats, and delayed oral bone loss by ovariectomy.

Mechano growth factor promotes the migration of bone marrow-derived mesenchymal stem cells / 中华物理医学与康复杂志

Objective To study the mechanism by which mechano growth factor (MGF) promotes the migration of mesenchymal stem cells (MSCs).Methods MSCs were isolated from Sprague-Dawley rats and treated with MGF at various concentrations.Western blotting was used to evaluate the expression of RhoA protein and its downstream p-MYPT,as well as p-FAK and t-FAK proteins related to focal adhesion kinase.The aim was to illustrate the effect of MGF in regulating the cytoskeleton of MSCs and the formation of focal adhesion.C3 toxin was used to inhibit RhoA activity and western blotting was used to examine the expression of p-MYPT,and the focal adhesion kinases p-FAK and t-FAK.Transwell assays were used to examine MSCs' migration ability,and immunofluorescence was conducted to examine the formation of F-actin cytoskeleton and focal adhesion.Results MGF can significantly promote the expression of MSCs' RhoA and its downstream protein p-MYPT.The effect is dose-dependent.The expression of RhoA and p-MYPT increased most significantly at 50 μM concentration.The ratio of p-FAK to t-FAK indicates that MGF can activate focal adhesion kinase and promote adhesion.C3 toxin significantly inhibited FAK activation.Transwell assays showed that MGF can significantly promote MSC migration,but pretreatment with C3 toxin inhibited it.The immunofluorescence results show that MGF can promote the rearrangement of MSCs' F-actin cytoskeleton and the formation of focal adhesion.C3 toxin disrupted MSCs cytoskeletons and decreased focal adhesion.Conclusion MGF promotes MSCs' migration through RhoA-and kinase-mediated cytoskeleton rearrangement and the formation of focal adhesion.

TNF-α，IL-1β induce mechano growth factor expression in human fibroblast-like synoviocytes through the PKA pathway / 医用生物力学

Objective To investigate the effects of inflammatory factors TNF-α, IL-1β, IL-6 on expression of mechano growth factor (MGF). Methods In the experimental group, TNF-α and IL-6 at concentration of 25, 50, 100 ng/mL, or IL-1β at concentration of 2.5, 5.0, 10 ng/mL were applied to fibroblast-like synoviocytes (FLSs) for 12 hours. The inhibitor groups were pretreated with PKA pathway inhibitor KT5720 at concentration of 1.0 mmol for 1 hour. The control group remained under the same culture condition as the experimental group, but without any growth factor. Real-time PCR was used to measure the gene expression of MGF. Results Treated with TNF-α at concentration of 25 ng/mL and IL-1β at concentration of 10 ng/mL, the MGF expression in FLSs was significantly increased (P＜0.05). IL-6 had no effect on MGF expression. A specific inhibitor of cAMP-dependent protein kinase, at concentration of 1.0 mmol significantly decreased the activation of MGF synthesis by TNF-α and IL-1β in FLSs (P＜0.05). Conclusions TNF-α at concentration of 25 ng/mL and IL-1β at concentration of 10 ng/mL significantly induce the MGF expression in FLSs, which activate MGF synthesis via the PKA pathway. This study is of significance in improving the application of MGF used in tissue repair area to make up the insufficient stress stimulation.

Effect of MGF-Ct24E and mechanical stimulation on gene expression of osteoblasts / 医用生物力学

Objective To identify the differentially expressed genes of osteoblasts under the stimulation of mechano growth factor E peptide( MGF-Ct24E) and mechanical stress by microarray analysis. Methods Primary osteoblasts were cultured in vitro, which were subjected to mechanical stimulation(with the mechanical strain of 12% and frequency of 0.5 Hz) and MGF-Ct24E treatment(50 mg/L), respectively. The gene expression profiles were analysed by cDNA microarrys and quantitative PCR was used to validate the microarray data. ResultsCompared with the control group, 1 866 genes were found to have differentially expressed in the mechanical loading group, in which 1 113 genes were up-regulated, while 753 genes were down-regulated. 1 178 genes were found to have differentially expressed in the MGF-Ct24E group, in which 796 genes were up-regulated and 382 genes were down-regulated. GO analysis suggested that the gene expression profile of MGF-Ct24E group was consistent with that of the mechanical loading group and differentially expressed genes were mainly involved in cell proliferation and differentiation, response to mechanical stress and mechaotransduction. ConclusionsThe microarray analysis showed that MGF-Ct24E treatment had similar effects with the mechanical loading on the gene expression of osteoblasts, which might provide a novel approach to study the usage of MGF-Ct24E for treating bone repair in the absence of mechanical stimulation.

Expression of mechano-growth factor in osteoblasts under mechanical stretch / 中华创伤杂志

Objective To analyze the protein expression and subcellular distribution of mechanogrowth factor (MGF) in ostcoblasts under stretch stimulation. Methods Cyclic stretching was applied to osteohlasts by a mechanical stretching device. The whole-cell proteins were extracted from controlled and stretched osteoblasts for detecting the protcin expression level of MGF by Western blot and observing the intracellular distribution of MGF by fluorescent immunocytological method. Results Western blot showed significant increase of expression of MGF in osteoblasts under stimulation of cyclic stretching. The level of protein was increased by four folds after 12-hour stretching of osteohlasts, and then declined sharply. Immunofluorescence analysis showed that MGF was mainly distributed in the nuclei of osteoblasts. ConcinsionsUnder the cyclic stimulation, the expression of MGF reaches a short period of peak in osteoblasts, which may be related to the injury of osteoblasts caused by stretching. MGF is mainly distributed in the nuclei of osteoblasts, indicating that MGF may contain nuclear localization signal and modulate the expression of relative genes.