The Study Of Underwater Low Intensity Pulsed Ultrasound Improved Senile Bone Loss In C57BL/6 Mice

ObjectivesOsteopenia is one of senile osteoporosis characterized by a"low-transformation"pattern,which osteogenic activity is significantly reduced while osteoclast activity remains at a certain level.Low intensity pulsed ultrasound?LIPUS?,as an effective physical therapy for mechanical signals of bone tissue,effectively avoids side effects of drug application,and has the advantages of non-traumatic,non-ionizing radiation and lower cost.Many studies have shown that LIPUS could promote fracture healing.As it was known that some biological mechanisms of fracture and bone loss are similar during intervention.Therefore,LIPUS may also be an important potential method to improve bone loss.Bone loss is a systemic disorder of bone metabolism.If LIPUS was directly used for local treatment,the efficacy is obviously limited.Better methods for treatment needs to be further explored.Therefore,this study intends to construct a underwater LIPUS method to treat the whole body of mice and explore the feasibility of underwater LIPUS device,and explore the effect of underwater ultrasonic treatment on bone mineral density?BMD?,bone turn-over biochemical indicators and biomechanical function of elderly mice.Methods:1.44 female C57BL/6 mice were selected according to the age of12¹4 months old and 20²3 months old,and then randomly divided into sham group and LIPUS group.Underwater LIPUS device was constructed and used for treatment in ultrasound group.The theoretical simulation of the underwater LIPUS device was carried out using COMSOL 5.3 software,and the sound pressure distribution map was drawn.The mice were bled by eyelids and serum calcium?Ca?and serum phosphorus?P?levels were measured using an automatic biochemical analyzer.Bone alkaline phosphatase?BALP?and?isomer of C-terminal telopeptide of type?collagen??-CTX?were detected by enzyme-linked immunosorbent assay?ELISA?.2.The number of spontaneous activities of mice in each group was recorded by autonomous activity recorder.BMD of lumbar vertebrae L_1⁵,left and right femurs of mice was measured by dual energy X-ray absorptiometry.The follow-up measurements of serum Ca,P,BALP and beta-CTX were the same as the first part.Estradiol?E₂?was determined by ELISA.The maximum load and displacement of femoral bending destruction were measured by three-point bending test.Results1.LIPUS underwater successfully penetrated the cortical bone and there was still a distribution of sound pressure in the medullary cavity.Compared with the 12¹4 months group,the serum Ca,P and?-CTX of20²3 months group were not significantly different?P>0.05?,while BALP levels were significantly lower?P<0.01?.Compared with the sham group,the serum Ca level of the mice in the ultrasound group was significantly decreased?P<0.01?,and the serum P level was significantly increased in both two age group?P<0.01?.What's more,the expression level of BALP in both the ultrasound group was significantly increased?P<0.01?,and the expression of?-CTX was only significantly decreased in the 20²3 months?P<0.05?.2.The number of spontaneous activities of mice aged 20²3 months was significantly less than that of mice aged 2³ months and 12¹4months?P<0.01?.There was no significant difference in the number of autonomous activities of at 0 d and 30 d after treatment between 12¹4months and 20²3 months of age?P>0.05?.The follow-up results of bone turn-over indicators showed that in the 12¹4 months old group,the blood Ca level in the LIPUS group at 30 d after LIPUS irradiation was still significantly lower than that in the control group?P<0.01?;The blood P level in the ultrasound group was significantly higher than that in the control group?P<0.05?;There was no significant difference in the serum BALP between the control group and the LIPUS group at 0 d after LIPUS?P>0.05?;And there was no difference in the serum?-CTX between the two groups?P>0.05?.In the group of 20²3 months old,the blood Ca in the LIPUS group at 30 d after LIPUS was significantly lower than that of the control group?P<0.01?,the blood P of the LIPUS group was significantly higher than that of the control group?P<0.05?;the serum BALP level was significantly lower than that of LIPUS group at 0 d after LIPUS?P<0.01?,slightly higher than that of the follow-up control group,but there was no statistical difference?P>0.05?.The level of serum?-CTX was lower than that of control group,and there was no significant difference between follow-up control group and LIPUS group at 0 d after LIPUS?P>0.05?.Estradiol in 20²3 months group were significantly lower than those in 2³ months group and 12¹4 months group?P<0.01?.In 12¹4 months old group,the level of estradiol increased significantly at 0d after LIPUS?P<0.05?.Follow-up at 30d after LIPUS showed that the level of estradiol in LIPUS group was significantly lower than that in LIPUS group at 0d after LIPUS?P<0.05?.In 20²3 months old group,the level of estradiol increased significantly at 0 d after LIPUS?P<0.05?.The level of estradiol in LIPUS group was significantly lower than that in LIPUS group at 0 d after LIPUS?P<0.05?,but there was no significant difference between LIPUS group and follow^up control group?P>0.05?.The trend was similar to that in 12¹4 months old group.BMD measurements of the L_1⁵,left and right femurs showed that BMD of the20²3 month old group was significantly lower than that of the control group aged 12¹4 month old at 0 d after LIPUS?P<0.05?.In the 12¹4month old group,the BMD of the L_1⁵,left and right femurs had no significant change?P>0.05?at 0 d after LIPUS,but the BMD at 30 d after LIPUS was significantly higher than that of the control group?P<0.05?.BMD results of left and right femurs showed that BMD of LIPUS group increased slightly at 0 d after LIPUS,but there was no significant difference?P>0.05?,while BMD of LIPUS group at 30 d after LIPUS was significantly higher than that of follow^up control group?P<0.05?.However,in BMD of L_1⁵ in 20²3 months old group,the follow-up data at 0 d after LIPUS and at 30 d after LIPUS were not significantly different from those of control group?P>0.05?.The trend of maximum load and maximum displacement was similar,which showed that the maximum load and maximum displacement of 20²3 months old group were significantly lower than those of 12¹4 month^old at 0d after LIPUS?P<0.05?.In 12¹4 months old group,the maximum load and displacement of LIPUS group were slightly higher than those of control group at 0 d after LIPUS,but there was no statistical difference?P>0.05?,but the maximum load and displacement of LIPUS group at 30 d after LIPUS were significantly higher than those of control group?P<0.05?.However,there was no significant difference in the maximum load and displacement between groups in the 20²3 months group?P>0.05?.ConclusionThe construction of underwater LIPUS device was feasible and it can effectively ameliorate the early biochemical bone turnover markers in aged C57BL6 mice.Underwater LIPUS can improve BMD,bone turnover markers and anti-fracture ability of C57BL/6 mice,but obviously it has better therapeutic effect on 12¹4 months old mice.