| The alveolar bone remodelling under mechanical stress is the biological basis of oral orthodontic tooth movement. Osteoblasts are one type of the important effector cells in the alveolar bone remodelling. Biomechanical effects of osteoblasts are crucial in the process of bone formation and remodelling. It is helpful that the biological characteristics and its signal transduction mechanism of osteoblasts subjected to mechanical stress are studied for understanding the mechanism of the alveolar bone remodelling in orthodontic treatment.The signal transduction mechanism of osteoblasts under mechanical stress is not very clear. m TOR plays an important role in the regulation of cell growth. And m TOR signal can be activated by mechanical stress in skeletal muscle. However, it is not clear whether cyclic mechanical stretch can activate osteoblastic m TOR signal and regulate osteoblastic proliferation, differentiation and energy metabolism through activation of m TOR signal.Through applying cyclic mechanical stretch to human osteoblast-like cells MG63(10%, 0.1Hz) in the present study, the effects of mechanical stretch on osteoblastic proliferation, differentiation and energy metabolism were observed and the regulatory mechanism of Akt/m TOR/p70s6 k signal pathways that mediated cellular responses to mechanical stretch was investigated. In addition, expression of Akt/m TOR/p70s6 k signaling pathways was observed in the periodontal ligament cells in the process of tooth movement through the orthodontic tooth movement experiment in rats to provide the basis for further study.Experiments one: Effects of cyclic mechanical stretch on proliferation and differentiation of osteoblast-like cells.Objective: To study effects of cyclic mechanical stretch on proliferation and differentiation of osteoblast-like cells. Methods: Cyclic mechanical stretch(10%, 0.1 Hz) was subjected to human osteoblast-like cells MG63. The s-phase cell fraction(SPF) and ALP activities were analysed by using flow cytometry and Alkaline Phosphatase Assay Kit. The expressions of ALP, OCN, runx2, COLI and P4HB(prolyl 4-hydroxylase, beta polypeptide) m RNA and ALP, runx2 and P4 HB proteins were analysed by using quantitative real-time polymerase chain reaction(q RT-PCR), immunofluorescence and western blot methods. The mineralized nodules were observed by using alizarin red S staining at the 5th and 6th week in the mineralized solution. Results: The SPF and ALP activities both increased at the 24 th hour of stretch loading(P<0.05). However, cyclic mechanical stretch had no effects on SPF and still could upregulate the ALP activity at the 3rd day in the mineralized solution(stretch loading for 8 hours per day). And cyclic mechanical stretch could upregulate the m RNA expression of ALP, OCN, runx2, COLI and P4HB(P<0.05), increase the protein expression of ALP, runx2 and P4HB(P<0.05) and promote the formation of mineralized nodules. Conclusions: When lower density in osteoblasts, cyclic mechanical stretch stimulated proliferation and upregulated the expression of osteogenesis-related m RNA and proteins to make preparations for subsequent osteogenetic differentiation in the respect of cell number and osteogenetic-related factors concentration. While density of osteoblasts and concentration of osteogenetic-related factors became a certain level, cyclic mechanical stretch promote differentiation instead of proliferation.Experiments two: Effects of cyclic mechanical stretch on energy metabolism of osteoblast-like cells.Objective: To study the effects of cyclic mechanical stretch on energy metabolism of osteoblast-like cells. Methods: Human osteoblast-like MG63 cells were subjected to cyclic mechanical stretch(10%, 0.1 Hz). Glucose consumption and lactate levels in the cell culture medium were determined using a Glucose/Lactate assay kit. The ATP concentrations were adetermined by using luminometer and r Luciferase/Luciferin(r L/L) reagent, and the expressions of ATP5 B, ATP5F1, ATP5 J, F1-ATPaseα, ENO1 and LDHA m RNA and ATP5 B and ATP5 J proteins were analysed by using q RT-PCR, immunofluorescence and western blot methods. Results: Cyclic mechanical stretch could upregulate the expressions of ATP5 B, ATP5F1, ATP5 J, F1-ATPaseα, ENO1 and LDHA m RNA(P<0.05), increase glucose consumption, lactate levels and ATP concentrations(P<0.05) and promote the expression of ATP5 B and ATP5 J proteins(P<0.05). Conclusions: Cyclic mechanical stretch could promote energy metabolism of osteoblast-like cells MG63 to provide energy for stretch-induced proliferation and differentiation through regulation of the key enzyme of energy metabolism.Experiments three: Effects of cyclic mechanical stretch on Akt/m TOR/p70s6 k signal pathway of osteoblast-like cells.Objective: To study the effects of cyclic mechanical stretch on Akt/m TOR/p70s6 k signal pathway of osteoblast-like cells. Methods: Human osteoblast-like MG63 cells were subjected to cyclic mechanical stretch(10%, 0.1 Hz). The semi-quantitative analysis of Akt, p-Akt(Thr308), p-Akt(Ser473), m TOR, p-m TOR(Ser2448), p70s6 k and p-p70s6k(Thr389) proteins were carried out by using immunofluorescence and western blot methods. Results: Cyclic mechanical stretch could upregulate the expression of p-Akt(Thr308), p-Akt(Ser473), p-m TOR(Ser2448) and p-p70S6K(Thr389)(P<0.05), but had no effects on Akt, m TOR, p70s6 k protein expression. Conclusions: Cyclic mechanical stretch could activate the Akt/m TOR/p70s6 k signal pathway of osteoblast-like cells MG63.Experiments four: The role of Akt/m TOR/p70s6 k signal pathway in proliferation and differentiation of osteoblast-like cells following cyclic mechanical stretchObjective: To investigate the role of Akt/m TOR/p70s6 k signal pathway in proliferation and differentiation of osteoblast-like cells following cyclic mechanical stretch. Methods: Rapamycin(m TOR inhibitor), wortmannin(Akt inhibitor) and DMSO(solvent control) were injected into medium before human osteoblast-like MG63 cells were subjected to cyclic mechanical stretch(10%, 0.1 Hz). The expressions of ALP, OCN, runx2, COLI and P4 HB m RNA and ALP, runx2, P4 HB, Akt, p-Akt(Thr308), p-Akt(Ser473), m TOR, p-m TOR(Ser2448), p70s6 k and p-p70s6k(Thr389) proteins were analysed by using q RT-PCR, immunofluorescence and western blot methods. The SPF and ALP activities were analysed by using flow cytometry and Alkaline Phosphatase Assay Kit. The mineralized nodules were observed by using alizarin red S staining at the 6th week in the mineralized solution. Results: Activation of Akt and m TOR signal under cyclic mechanical stretch could be suppressed by wortmannin(P<0.05). However, activation of m TOR but not Akt signal under cyclic mechanical stretch could be inhibited by rapamycin(P<0.05). Higher proportion of the SPF of osteoblast-like cells could be decreased by wortmannin and rapamycin under cyclic mechanical stretch(P<0.05). The upregulation of m RNA expressions of ALP, OCN, runx2, COLI and P4 HB can be inhibited by rapamycin(P<0.05), but except for ALP, the upregulation of other m RNA expressions can not be inhibited by wortmannin under cyclic mechanical stretch. And increasing of ALP, runx2 and P4 HB proteins, ALP activies and mineralized nodules under cyclic mechanical stretch can be suppressed by rapamycin but not wortmannin. Conclusions: Cyclic mechanical stretch could stimulate the proliferation of osteoblast-like cells MG63 through the activation of Akt/m TOR/p70s6 k signal pathway. However, cyclic mechanical stretch could promote the differentiation of osteoblast-like cells MG63 denpendent of m TOR signal but independent of Akt signal.Experiments five: The role of Akt/mTOR/p70s6 k signal pathway in energy metabolism of osteoblast-like cells following cyclic mechanical stretchObjective: To investigate the role of Akt/m TOR/p70s6 k signal pathway in energy metabolism of osteoblast-like cells following cyclic mechanical stretch. Methods: Rapamycin(m TOR inhibitor), wortmannin(Akt inhibitor) and DMSO(solvent control) were injected into medium before human osteoblast-like MG63 cells were subjected to cyclic mechanical stretch(10%, 0.1 Hz). Glucose consumption and lactate levels in the cell culture medium were determined using a Glucose/Lactate assay kit. The ATP concentrations were determined by using luminometer and r Luciferase/Luciferin(r L/L) reagent, and the expressions of ATP5 B, ATP5F1, ATP5 J, F1-ATPaseα, ENO1 and LDHA m RNA and ATP5 B and ATP5 J proteins were analysed by using q RT-PCR, immunofluorescence and western blot methods. Results: The upregulation of glucose consumption, lactate levels, ATP concentrations, and ATP5 B and ATP5 J proteins under cyclic mechanical stretch can be suppressed by wortmannin and rapamycin(P<0.05). Except for ENO1, the upregulation of m RNA expressions of ATP5 B, ATP5F1, ATP5 J, F1-ATPaseα and LDHA can be inhibited by wortmannin and rapamycin(P<0.05). Conclusions: Cyclic mechanical stretch could promote the energy metabolism of osteoblast-like cells MG63 through the activation of Akt/m TOR/p70s6 k signal pathway.Experiments six: Effects of orthodontic tooth movement on Akt/m TOR/p70s6 k signal pathway in the periodontal ligament cells of rats.Objective: To observe effects of orthodontic tooth movement on Akt/m TOR/p70s6 k signal pathway in the periodontal ligament cells of rats. Methods: Fifteen SD rats(age, 8 weeks old; weight, 227±3.4g) were randomly divided into experimental groups(stretch) and the control group(un-stretch). In the Exp groups, a nickel-titanium closing coil spring was linked between the first molars of the right side of the maxillary dentition and maxillary central incisor using light-cured adhesive. Through regulating closing coil spring length, the first molars were subjected to constant tensile force of 30 g. The control rats were subjected to the same procedures but without stretching nickel-titanium closing coil spring. The rats were sacrificed and the right maxillary first molars, together with alveolar bone, were excised and immediately fixed at the end of the 1st, 3rd, 9th and 15 th day after the beginning of the experiment. The tissue blocks were decalcified, dehydrated, paraffin-embedded and then cut into sections and immunohistochemical staining were applied to observe the Akt, m TOR and p70s6 k protein expression of the periodontal ligament cells in the tensile and compressive regions of periodontal ligament. Results: The gap began to appear between the first and second molars of the right side of the maxillary at the 3rd day, and the gap became bigger at the 9th day(P<0.05), and there were no significant differences at 15 th and 9th days. The Akt, m TOR and p70s6 k protein of periodontal ligament cells increased in the process of tooth movement along with time. The Akt protein expression reached its peak at the 3rd day in the tensile regions and at the 9th day in the compressive regions, and subsequently descend to some extent, but still was higher than the control group(P<0.05). m TOR and p70s6 k protein expression reached its peak at the 9th day in the tensile and compressive regions, then declined, but were still higher than the control group(P<0.05). Conclusions: Expression of Akt/m TOR/p70s6 k of periodontal ligament cells both increased in the tensile and compressive regions of periodontal ligament in the process of orthodontic tooth movement in rats. |
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