| PURPOSE:In this work the effect of low-intensity pulsed ultrasound (LIPUS) and extracorporeal shock wave (ESW) to osteoporosis were investigated. Ultrasound and shock wave of different parameters (frequency and intensity) were applied to MC3T3-E1cell. The cell proliferations, differentiation, and mineralization were determined by CCK-8method, alkaline phosphatase (ALP) detection kit, and alizarin red staining, respectively. Parameters of stimulation were selected then. Mice osteoporosis model caused by ovariectomy was established as subject to verify effectiveness of LIPUS and ESW to osteoporosis in vivo. The results of bone mineral density and biomechanics showed that low-intensity pulsed ultrasound and extracorporeal shock wave are efficacious methods for osteoporosis physical therapy. Pathological section of organs suggested they are of considerable biosafety. Mechanical signals may be a potential way of prevention and treatment of osteoporosis.METHODS:1.LIPUS cell experimentMouse osteoblastic cell line MC3T3-E1was cultured in a-MEM mediums placed in incubator at37℃,5%CO2. The cell medium was exchanged every3days. Cells were subcultured every3or4days. Logarithmic cells were incubated onto96-well plate and12-well plate for densities as2×103cell/well and2×104cell/well. Experimental subjects were divided by the intensity for4groups:30mW/cm2,40mW/cm2,50mW/cm2, and blank control (CON). On96-well plate each condition corresponded with6parallel wells and on12-well plate each condition corresponded with4parallel wells. Cell proliferation effect was detected at the end of2nd,4th, and6th day. ALP activity was detected at the end of10th day. Mineralization effect was detected at the end of14day.2. ESW cell experimentCells were cultured in the same way in LIPUS experiment. See table1for grouping and parameters. Mice in each group received stimulation for20min per day. Cell proliferation effect was detected at the end of2nd,4th, and6th day. ALP activity was detected at the end of10th day. Mineralization effect was detected at the end of14th day.3.LIPUS and ESW animal experimentHealthy female BalB/C mice (week8, SPF grade) received bilateral ovariectomy (OVX) so mice osteoporosis model was established. Mice were divided into4groups:blank control (CON), estrogen control group (ESG),low-intensity pulsed ultrasound (LIPUS), extracorporeal shock wave group (ESW). Each group contained6mice. Optimal parameter selected by cell experiment was applied to LIPUS, which was1.5MHz-40Mw/cm2, applied every5min. Optimal parameter selected by cell experiment was applied to SEW, which was10.1Hz-1.12g. ESG received conjugated estrogens tablets solution gavage at a dosage of0.39mg/kg. Mice in all groups received stimulation5days per week. Stimulation last for8weeks. Bone mineral density of lumbar was measured using dual energy x-ray absorptiometry after intervention cycle ended. Lumbar vertebra of all groups was used for osseous tissue biomechanics detection and histologicalsection and Hematoxylin-Eosin (HE) staining, to acquire maximum load (Lmax,N), maximum displacement (Dmax,mm), and osseous tissue morphology image. Main organs of mice of LIPUS and ESW were used for histologicalsection and HE stain, to observe under microscope for safety evaluation.RESULTS:1. LIPUS cell experiment1.1Proliferation:30mW/cm2and40mW/cm2intensities pulsed ultrasound had accelerated cell proliferation (P<0.01), however,50mW/cm2intensity pulsed ultrasound caused inhibition (P<0.01) at all3different frequencies.1.2Differentiation:at the end of10th day under the function of pulsed ultrasonication, in contrast to CON, ALP activity increased in MC3T3-E1cells of subgroups of1.1MHz-50mW/cm2,1.5MHz-30mW/cm2,1.1MHz-40mW/cm2, and1.7MHz-30mW/cm2(P<0.05).1.3Mineralization:at the end of13th day under the function of pulsed ultrasonication, in contrast to control group, obvious mineralization nodules were observed in MC3T3-E1cells of subgroups of1.5MHz-30mW/cm2,1.5MHz-30mW/cm2,1.7MHz40mW/cm2, and1.7MHz-40mW/cm2.2. ESW cell experiment2.1Proliferation:only amplification of optical density values (OD) in subgroups of7.3Hz-0.58g and10.1Hz-0.56g was statistically significant. Extent of proliferation in other subgroups was unconspicuous.2.2Differentiation:in contrast to CON, extent of differentiation increased in all ESW subgroups to varying degrees. Amplification in subgroup of3.3Hz-0.12g, all3subgroups of7.3Hz, and all3subgroups of10.1Hz was statistically significant (P<0.05). ALP activitysignificantly increased in subgroups of3.3Hz-0.18g and10.1Hz-1.68g. Compared to CON, no difference was observed in other subgroups.2.3Mineralization:in contrast to CON, obvious mineralization nodules were observed in subgroups of3.3Hz-0.12g,7.3Hz-0.58g,7.3Hz-0.87g,10.1Hz-1.12g, and10.1Hz-1.68g, of which subgroup of10.1Hz-1.12g had significantly bettermineralizationeffect than any other subgroups.3.LIPUS and ESW animal experiment3.1Modeling evaluation3.1.1Estradiol:postoperative estradiol content in serum of mice was28.38pg/ml, which was significantly lower than preoperative data (64.72pg/ml)(P<0.05).3.1.2Bone mineral density:postoperative area of high bone mineral density (green) in vertebral region of mice significantly decreased, compare to preoperative data. Area of high bone mineral density (red) increased correspondingly. Quantitative analysis result from accompanying software of instrument revealed that both BMD and bone mass decreased significantly after operation.3.2Assessment of treatment effect3.2.1Bone mineral density:area of high bone mineral density modestly increased in ESG and LIPUS. No obvious effect was observed in ESW. Detailed data demonstrated that bone mineral density of both LIPUS and ESW increased, in contrast to CON (P<0.05). Increase of bone mass only occurred in ESW and no obvious difference was observed in other groups.3.2.2Lmax and Dmax:both Lmax and Dmax increased in ESG, LIPUS, and ESW, in contrast to CON (P<0.05). ESW was the group produced the best result.3.2.3Pathological section of organs:no pathological change was observed in tissues, no haemorrhage was observed, the cellular structure remained intact.CONCLUSIONS:1. Low-intensity pulsed ultrasound effectively accelerated proliferation, differentiation, and mineralization levels in MC3T3-E1cells. Extracorporeal shock wave effectively accelerated differentiation and mineralization levels in MC3T3-E1cells. LIPUS and ESW are of positive significance at the cellular level.2. LIPUS and ESW can also effectively enhance osteoporotic mice’ bone mineral density, increase their bone strength and bone stiffness, and retard or inhibit loss of bone density. When applied to animals they produce favorable application effect.3. The modeling method adopted in this work, ovariectomy, produced expected effect. It has been proved to be an effective modeling approach to establish animal model of osteoporosis. 4. Parameters and dosages of ultrasound and shock wave adopted in this work were of favorable biosafety. The optimal parameter for LIPUS was1.5MHz-40mW/cm2. The optimal parameter for ESW was10.1Hz-1.12g.Low-intensity pulsed ultrasound and extracorporeal shock wave are efficacious methods for osteoporosis physical therapy. They are of considerable biosafety. |
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