| Research background and objective:At present,osteoporosis is one of the main diseases that endanger the life and health of the elderly.In addition,delayed union and nonunion of fracture are still the difficulties in orthopedic treatment.In recent years,LMHFV characterized by a non-invasive method,less side effects,low cost,good compliance and other characteristics,have become an effective way in the prevention and treatment of osteoporosis and nonunion of fracture.We have preliminary data in the lab demonstrating:in biomechanical research,intermittent LMHFV loading can be more effective resist osteoporosis induced by castration or suspension.In vivo study of sheep also found that LMHFV can significantly promote fracture healing,and the effect of intermittent LMHFV loading is better.In the study of cell and molecular biology,we found that osteoblasts can sense the loading of LMHFV through primary cilia,thus up regulating the expression of osteoblast related proteins such as OCN,ALP and COL-1.However,the mechanism by which intermittent LMHFV loading can promote bone formation more effectively remains unclear.The research on this subject has not been reported at home and abroad.This research will take this as the breakthrough point,to study whether intermittent LMHFV loading is better than persistent LMHFV loading in promoting osteoblast differentiation and mineralization,and whether primary cilia play an important role in it.This will further explain the mechanism of mechanical stress such as LMHFV on osteoblast function,provide clues for bone tissue engineering design,optimize the application of LMHFV in the treatment of osteoporosis and nonunion of fracture,and provide theoretical basis and guidance for it.Methods:In this experiment,to study the effects of intermittent and persistent LMHFV loading on the proliferation,differentiation and mineralization of MC3T3-E1 cells,the cells were randomly divided into three groups:static control group,persistent LMHFV group and intermittent LMHFV group.LMHFV with vibration frequency of 35Hz and vibration intensity of 0.25g was applied to persistent LMHFV group for 5 or 12consecutive days,30min a day,while LMHFV with the same duration,intensity and frequency was applied to intermittent LMHFV group every other day.The period of 5days was used to study osteogenic differentiation,while the period of 12 days was used to study osteogenic mineralization.To study the role of primary cilia in promoting osteogenic differentiation and mineralization of MC3T3-E1 cells under intermittent and persistent LMHFV loading,cells were randomly divided into four groups:persistent LMHFV group,persistent LMHFV with chloral hydrate group,intermittent LMHFV group and intermittent LMHFV with chloral hydrate group.Among them,the persistent LMHFV with chloral hydrate group and intermittent LMHFV with chloral hydrate group were interfered by chloral hydrate at a concentration of 4mM.The vibration loading method is the same as the above.To study the effect of single LMHFV loading on the morphology and incidence rate of primary cilia of osteoblasts,the cells were randomly divided into four groups:static control group,1 hour after LMHFV loading group,24hour after LMHFV loading group and 48 hour after LMHFV loading group.LMHFV with vibration frequency of 35Hz and vibration intensity of 0.25g was applied to each group except the control group for 30min,and the properties of primary cilia were observed and quantitatively analyzed under laser scanning confocal microscope after 1h,24h and 48h,respectively.To study the effect of intermittent and persistent LMHFV loading on the morphology and incidence rate of primary cilia of osteoblasts,cells were randomly divided into intermittent LMHFV group and persistent LMHFV group,and then each group was divided into 1 hour before the first day of vibration group,1 hour after the first day of vibration group,1 hour before the second day of vibration group and 1 hour before the third day of vibration group.LMHFV was continuously applied for 30min a day with vibration frequency of 35Hz and vibration intensity of 0.25g in persistent LMHFV group,while LMHFV with the same duration,intensity and frequency was applied in intermittent LMHFV group every other day.Laser scanning confocal microscopy,ELISA,immunohistochemical staining and Western blot were used for qualitative and quantitative analysis.Results:To study the effects of intermittent and persistent LMHFV loading on the proliferation,differentiation and mineralization of MC3T3-E1 cells,we found that there was no significant difference in total DNA content among the static control group,intermittent LMHFV group and persistent LMHFV group(P>0.05).Compared with the ALP staining area of 3.75±0.41cm~2 in the static control group,the persistent LMHFV group was 6.67±0.24cm~2(P<0.01),and the intermittent LMHFV group was7.74±0.33cm~2(P<0.01).Compared with the control group,the expression of OCN and COL-1 in intermittent and persistent LMHFV group was significantly higher(P<0.01).The area of calcium nodule staining in the persistent LMHFV group was 4.82±0.47cm~2and that in the intermittent LMHFV group was 6.62±0.61cm~2,which was significantly higher than that in the static control group(2.14±0.22cm~2)(P<0.01).Compared with the persistent LMHFV group,the staining area of ALP in intermittent LMHFV group was significantly deepened(P<0.05),the expression of OCN was significantly increased(P<0.05),the expression of COL-1 was also significantly increased(P<0.01),and the staining area of calcium nodules was also significantly increased(P<0.01).To study the role of primary cilia in promoting osteogenic differentiation and mineralization of MC3T3-E1 cells under intermittent and persistent LMHFV loading,we found that the ALP staining area of intermittent LMHFV+CH group was 3.72±0.47cm~2 and that of persistent LMHFV+CH group was 3.39±0.34cm~2,and the staining area of calcium nodules in intermittent LMHFV+CH group was 2.08±0.24cm~2,and that in persistent LMHFV+CH group was 1.99±0.17cm~2,which was significantly lower than that in the corresponding without chloral hydrate group(P<0.01).This phenomenon is also found in OCN and COL-1 protein expression in intermittent LMHFV+CH group and persistent LMHFV+CH group.And there were no significant differences in ALP staining area,OCN,COL-1 protein expression and calcium nodule staining area between the two groups(P>0.05).To study the effect of single LMHFV loading on the morphology and incidence rate of primary cilia of osteoblasts,we found that the incidence rate of primary cilia was 64.1±4.2%in the static control group and 34.6±3.6%one hour after LMHFV loading(P<0.01).Compared with 1 hour after LMHFV loading group,the incidence rate of primary cilia was 49.2±3.9%24 hours after LMHFV loading(P<0.01).48 hours after LMHFV loading,the incidence rate of primary cilia was 62.8±4.4%,which was close to that of the control group(P>0.05).The length of primary cilia was 3.07±0.82μm in the static control group and 0.93±0.23μm one hour after LMHFV loading(P<0.01).Compared with 1 hour after LMHFV loading group,the length of primary cilia was1.9±0.27μm 24 hours after LMHFV loading(P<0.01).48 hours after LMHFV loading,the length of primary cilia was 2.84±0.84μm,which was close to that of the control group(P>0.05).To study the effect of intermittent and persistent LMHFV loading on the morphology and incidence rate of primary cilia,we found that one hour after LMHFV loading on the first day of vibration cycle,the incidence rate and length of primary cilia in intermittent LMHFV group and persistent LMHFV group decreased significantly,but there was no significant difference between the two groups(P>0.05).One hour before LMHFV loading on the second day of vibration cycle,compared with the previous group,the incidence rate and length of primary cilia increased in intermittent LMHFV group and persistent LMHFV group,but there was still no significant difference between the two groups(P>0.05).Until 1 hour before LMHFV loading on the 3rd day of vibration cycle,compared with the incidence rate of primary cilia in the 1 hour before LMHFV loading on the first day of vibration cycle(63.52±3.9%),the incidence rate of primary cilia in intermittent LMHFV group was 63.47±2.6%(P>0.05).However,compared with intermittent LMHFV group,the incidence rate of primary cilia in persistent LMHFV group was significantly reduced to 27.7±1.8%(P<0.01).Compared with the length of primary cilia in the 1 hour before LMHFV loading on the first day of vibration cycle(3.06±0.59μm),the length of primary cilia in intermittent LMHFV group was 2.96±0.47μm(P>0.05).However,compared with intermittent LMHFV group,the length of primary cilia in persistent LMHFV group was significantly reduced to0.87±0.2μm(P<0.01).Conclusion:The results show that intermittent LMHFV and persistent LMHFV can sense and conduct through primary cilia of osteoblasts,up regulate the expression of osteogenic related proteins and promote the mineralization of osteoblasts,and intermittent LMHFV is better than persistent LMHFV.The removal of primary cilia can counteract the difference between intermittent and persistent LMHFV in promoting osteogenesis.Through the changes of morphology and incidence rate of primary cilia under intermittent and persistent LMHFV loading,we found that LMHFV can cause primary cilia consumption.Persistent LMHFV loading will lead to primary cilia fatigue,while intermittent LMHFV loading can make primary cilia recover to a certain extent,and primary cilia state is better,so its osteogenic effect is better.Our findings provide new insights into the potential mechanism of LMHFV induced osteogenesis,and optimize the loading scheme of LMHFV in the treatment of osteoporosis and nonunion of fracture. |
