The Initial Biological Responses To Vibration Stress And The Mechanotransduction In Osteoblasts In Vitro

Backgrounds:As a systemic skeletal disease,osteoporosis(OP) is characterized by bone loss, degradation of bone tissue microstructure,and the resulting in increasing of bone brittleness and easy fracture.One of OP's major complication is OP fractures,a common disease which is harmful to the health of the elderly,Especially in the postmenopausal women.Movement is one of the important ways for the prevention and treatment of OP,according to correlated investigation:with regard to the elderly with OP and the postmenopausal women,physical exercise and activities can help to maintain bone mineral density and reduce the chanciness of OP.People have already recognized that mechanical factors play an important role in the process and development of OP.The mechanical vibration is one of mechanics function forms. some researches indicate that vibration is a favorable promotion to bone formation and bone remodeling,Vibration Stress(VS) is good to bone maturation and improvement,but vibration strength and frequency has influence on the bone formation,at present,there are significant different conclusions because of different experimental methods and models on the research field in the vibration frequency at home and abroad,and the overall frequency is different by range from 1Hz to 100Hz.Mechanical load can inhibit bone resorption and increase bone formation and bone cells can transform mechanical signal into cellular biochemistry synthesis reaction,which has a broad application prospects in the treatment of OP and the prevention of bone loss in the elderly.However,the mechanism of mechanical signal transduction remains unclear.Objectives:1.To speculate the form of the stress loaded to the osteoblasts(OBs) during vibrating.2.To approach the suitable vibration frequency for promoting the proliferation and differentiation of OBs.3.To approach mechanotransduction of VS affecting OBs function at earlier stage.Methods:1.VS loading system was designed and made;Sprague-Dawley suckling mice cranial bones OBs were isolated and cultured by enzyme digestion,identified through morphology,specific enzyme staining and mineralization tubercle alizarin bordeaux staining:To estabilsh VS loading cell modes in vitro,to observe the movement situation of the liquid in culture dish and the appearance growth situation of cells during vibrating.2.Select OBs of the sixth generation for experiments,divide them into 7 groups at random:blank control group(group A),NaF positive control group(group B)and VS loading group(group C to G):vibration groups' corresponding frequency respectively are 3～10Hz,15～30Hz,25～45Hz,50～80Hz,80～100Hz,other vibration conditions was identical;cell culture time of each group was identical, group A and B didn't vibrate,group C to G load with VS according to request; compare cell cycle,cell reproductive activity and ALP activitiy between each group by One-way ANOVA method.3.Select OBs of the third generation for experiments,divide them into 4 groups at random:blank control group(group A),Verapamil group(group B),TTX group (group C),and Verapamil+TTX group(group D),each group respectively divide into 0min group and 30min group on the basis of vibration time,8 groups in all;Use different vibration frequency from 15Hz to 45Hz to interfere in specimens according to experiment design;Compare the cell membrane potential and intracellular Ca²⁺ between each group by One-way ANOVA,Independent-Samples T Test and Factor Analysis method.4.Select OBs of the third generation for experiments,divide them into 4 groups at random:blank control group(group A),blocking agent group(respec tively TTX and Verapamil,group B),vibration group(group C),and vibration+ blocking agent group(group D).Use different vibration frequency from 15Hz to 45Hz to interfere in specimens according to experiment design.Compare the cell reproductive activity and ALP activity between each group by One-way ANOVA method.5.Select OBs of the third generation for experiments,divide them into 2 groups at random:control group and vibration group.Each group respectively divide into 0min group,20min group,40min group and 60min group on the basis of vibration time,8 groups in all.Use different vibration frequency from 15Hz to 45Hz to interfere in specimens according to experiment design.Compare the cell secretion NO activity between each group by One-way ANOVA,Independent-Samples T Test and Factor Analysis method.Results:1.VS loading system well runs,vibration parameters output stably;OBs grow sticking to plates in fusiform shape and triangle under inverted microscope, positive in mineralization tubercle alizarin bordeaux staining and positive in ALP staining,identified to be OBs;Before vibrating,the surface of liquid stands still in culture dish;In the course of vibration,there were small fluctuant waves in the center of the surface of the liquid toward periphery,the shape and velocity of the fluctuant waves changed with various vibration frequency,and no deciduous cells and apoptosis cells could be seen under the stimulus of VS;cells grown in certain direction;VS loading group cells grown more quickly than blank control group, therefore VS loading OBs mode is effective.2.Frequency from 15Hz to 30Hz and 25Hz to 45Hz could induce OBs cycle,promote cells proliferation,and strengthen ALP activity;Frequency from 80Hz to 100Hz and 3Hz to 10Hz could suppress OBs cycle;Frequency from 80Hz to 100Hz could suppress cells proliferation;Frequency 50Hz to 80Hz and 80Hz to 100Hz could suppress ALP activity,statistical significance in discrepancy above(P＜0.01).3.(1) Significant deviation in statistics by Factor Analysis Interaction(F=30.646, P=0.000);Significant deviation in statistics(F=14.086,P=0.000) from different time groups between each group(A,B,C,D);Membrane potential fluorescence value is A group(286.32 in mean),B group(262.01 in mean),C group(284.38 in mean) and D group(255.45 in mean) by decrease;(2) VS loading OBs for 0min,cell membrane potential fluorescence intensity was at the same baseline level in each group,not significant deviation in statistics (P=0.081);VS loading OBs for 30min,cell membrane potential fluorescence intensity heightened to different extend,significant deviation in statistics (P=0.000);not significant deviation in statistics(P=0.444) between A2 and C2 by SNK method;(3) Cell membrane potential has significant deviation in statistics between 0min and 30min(P＜0.05);fluorescence intensity increases by 48.69%,30.00%,59.29%, and 27.30%according to A group,B group,C group and D group.4.(1) Significant deviation in statistics by Factor Analysis Interaction(F=36.841, P=0.000):Significant deviation in statistics(F=14.086,P=0.000) from different time groups between each group(A,B,C,D);intracellular Ca²⁺ fluorescence intensity value is A group(250.75 in mean),B group(233.12 in mean),C group (237.66 in mean) and D group(220.71 in mean) by decrease;(2) VS loading OBs for 0min,intracellular Ca²⁺ fluorescence intensity was significant deviation between each group(P=0.000);not significant deviation in statistics(P=0.576) between C₁ and D₁ by SNK method;VS loading OBs for 30min,intracellular Ca²⁺ fluorescence intensity heightened to different extend, significant deviation in statistics(P=0.000);not significant deviation in statistics (P=0.320) between B₂ and D₂ by SNK method; (3) Ca²⁺ fluorescence intensity has significant deviation between 0min and 30min for each group(P＜0.05);fluorescence intensity increases by 29.71%,13.90%, 32.46%and 13.73%according to A group,B group,C group and D group.5.(1)The effect of VS promoting OBs reproductive activity could not besuppressed by Na⁺ channel blocker TTX(P=0.630),but the effect of VS promoting OBs ALP activity could be suppressed by TTX(P=0.004);(2)The effect of VS promoting OBs reproductive activity and strengthening ALP activity could be suppressed by L-Ca²⁺ channel blocker Verapamil(P=0.000).6.(1) Significant deviation in statistics by Factor Analysis Interaction(F=4.076, P=0.013);(2) Not significant deviation between 4 time interval for NO secretory volume in blank control group during 60min(P=0.097);(3)Significant deviation between each time interval for NO secretory volume in vibration group during 60min(P=0.000);Compare with 0min group,the P value was 0.043,0.012,and 0.000 for 20min group,40min group and 60min group.Compare with 20min,not significant increasing for 40min group(P=0.555), but significant increasing for 60min group(P=0.010);the production of NO increases slowly in 40min at vibration group(P=0.012),but increase significantly after 40min(P=0.000);(4) Comprare with vibration group and blank control group between each time interval,at 0min,NO secretory volume of blank control group increase a little more than that of vibration group;at 20min and 40min,NO secretory volume of blank control group decrease a little more than that of vibration group,not significant deviation;at 60min,NO secretory volume of blank control group decrease more significantly than vibration group(P=0.007).Conclusions:1.During the course of VS loading,the stress form which OBs receives may be the fluid shearing stress(FSS). 2.Various vibration frequency influenced on cell cycle,reproductive activity and ALP activity of OBs;the suitable frequency range which promoted OBs proliferation and differentiation is 15Hz to 45Hz.3.VS active voltage dependent L-Ca²⁺ channel was one of the causes for the increasing of depolarization of OBs membrane potential,intracellular Ca²⁺ concentration,the increasing of intracellular Ca²⁺ concentration and NO activity could promote cells function and participate in the mechanotransduction in OBs in vitro at earlier stage.