| Objectives:Recently, as the life span of human being increasing a lot, China hasentered an aging society. In this context, the incidence of age-related disorders presents a gradual upward trend, especially in orthopedi-cs in which the incidence of degenerative disorders and osteoporosis disorders has been rising gradually. Knee osteoarthritis is the most common one among degenerative disorders and with the development of medical technology and popularization of health education, elderly pa-tients who suffered from this disease are willing to improve living quality, so the number of patients who received total knee arthroplasty to treat knee osteoarthritis increased dramatically. However, with large-scale application of this surgery, cases of loosening of prosthesis resulting from periprosthetical osteolysis after artificial joint replacement increased greatly. Because of this, the patient had to receive the second operation which not only bring extra pain and financial burden to them, but also increase medical risks. At pre-sent, many reasons can explain periprosthetical osteolysis, such as low toxicity infection, stress mechanism and particle mechanism. For the latter two mechanisms, from the perspective of western medicine, anti-osteoporosis therapy was used to regulate bone metabolism which has been proved to be effective. In last two decades, either on basic or clinical research, Chinese medicine has done a lot of work on anti-osteoporosis study. However, rare research has been performed on prosthetical osteolysis from the perspective of Chinese medicine and the application of Chinese medicine to treat bone mass loss after artificial joint replacement is still in blank field. So, the present study aimed at investigating the effect of Bushen qiangjin capsule on bone absorption around the artificial knee prosthesis and testing whether the capsule is effective by measuring the change of bone density around prosthesis, OPG and RANKL before and after treatment, and the related mechanism are also discussed in this paper.Methods and Materials一、 Clinical ObservationFrom February,2013 to February,2014, subjects who had knee osteoarthritis and received total knee arthroplasty were recruited from Guangdong second traditional Chinese medicine hospital. All subjects were given informed consent. A randomized double blind control study was conducted. As a result, 27 subjects were assigned to treatment group and 25 subjects were assigned to control group. All subjects received single knee arthroplasty and PS prostheses from LINK Company were used in the operation. All prostheses were fixed with bone cement. All subjects received the same rehabilitation methods and HSS score were performed after operation. In control group, Caltrate was continuously administered (0.6 g each time, two times per day) for 26 weeks postoperatively. In treatment group, Bushen qiangjin capsule (once a time, three times per day) was continuously administered for 26 weeks besides Caltrate postoperatively. Four region of interest (ROI) were divided around femur prosthesis, which are ROI1, R0I2, ROI3 and ROI4 respectively. Three ROI were divided around tibia, which are ROI1, ROI2 and ROI3 respectively. Dual-energy X-ray absorptiometry was used to observe the bone density in the above regions. One-way analysis of variance was used to analyze HSS score in one-week, three-month, six-month and twelve-month after operation in the two groups.二、 Clinical Experimental StudyThe morning fasting venous blood of all subjects was obtained to detect OPG and RANKL by ELISA and the data of two-week, three-month and six-month were recorded. PS prostheses from LINK Company (Germany) were used in total knee arthroplasty and all prostheses were fixed with bone cement. All subjects received the same rehabilitation methods and HSS score was conducted in the following 12 months after operation. Four ROI were divided around femur prosthesis, which are ROI1, ROI2, ROI3 and ROI4 respectively. Three ROI were divided around tibia, which are ROI1, ROI2 and ROI3 respectively. Dual-energy X-ray absorptiometry was used to observe the bone density in the above regions. One-way analysis of variance was used to analyze HSS score in one-week, three-month, six-month and twelve-month after operation in the two groups.三、Experimental Study Forty male mice were divided into five groups to conduct the experiment. The first group was the control group. The second was the model group. The third was he low-dosage bushen qingjin capsule group. The forth was the middle-dosage bushen qiangjin capsule group. The fifth was the high-dosage bushen qiangjin capsule group. Mice in the control group received sham-operation. For the other four groups, after opening skull, PMMA particle was smeared in the mice skull plates. Next, the mice were given by gavage with different dosage and fed with normal diet. Finally, the mice were executed and the skull tissue was extracted to calculate area of lacuna skull. By using PCR, mRNA expression of total DNA of bone tissue in middle skull was analyzed. Meanwhile, OPG and RANKL protein expression were detected with Western blotting method.Results一、 Clinical Observational Study(一) HSS ScoreResults showed that no subject complained any infection or prosthesis loosening in the 12-month follow-up, four subjects complained slight knee pain without interference of normal activity.HSS score:No significant statistical difference existed between treatment group and control group in one week and twelve month after operation (P> 0.05). Significant statistical difference existed between treatment group and control group in three month and six month after operation (P<0.01, P< 0.05); Significant statistical difference existed between treatment group and control group in the week before and after operation (P<0.01);No significant statistical difference existed in the treatment group between the six month and twelve month after operation (P>0.05);Significant statistical difference existed in the control group between the six and twelve month after operation (P<0.01).(二) Bone Density Observational Study1. Bone density change in treatment group and control group in ROI 1 Treatment group:compared with the week after operation, bone density in the year after operation deceased 20.32% (P<0.01);bone density in 3 month after operation decreased 11.05% (P<0.01); compared with 3 month after operation, no statistical difference existed in the bone density change in 6 month after operation (P>0.05);compared with 6 month after operation, no statistical difference existed in the bone density change in the year after operation (P>0.05).Control group:compared with the week after operation, bone density in the year after operation deceased 26.26% (P<0.01);bone density in 3 month after operation decreased 11.01% (P<0.05); compared with 3 month after operation, statistical difference existed in the bone density change in 6 month after operation (P<0.05);compared with 6 month after operation, no statistical difference existed in the bone density change in the year after operation (P>0.05)No statistical difference existed in the bone density change in the week after operation between the treatment and control group (P>0.05); Statistical difference existed in the bone density change in the year after operation between the treatment and control group (P<0.05);2. Bone density change in treatment group and control group in ROI 2 Treatment group:compared with the week after operation, bone density in the year after operation deceased 19.30% (P<0.01);bone density in 3 month after operation decreased 10.31% (P<0.01); compared with 3 month after operation, no statistical difference existed in the bone density change in 6 month after operation (P>0.05);compared with 6 month after operation, no statistical difference existed in the bone density change in the year after operation (P>0.05).Control group:compared with the week after operation, bone density in one year after operation deceased 25.18% (P<0.01);bone density in 3 month after operation decreased 9.96% (P<0.01); compared with 3 month after operation, statistical difference existed in the bone density change in 6 month after operation (P<0.05) and compared with the week after operation, bone density decreased 18.33%;compared with 6 month after operation, no statistical difference existed in the bone density change in one year after operation (P>0.05).No statistical difference existed in the bone density change in the week after operation between the treatment and control group (P>0.05); Statistical difference existed in the bone density change in the year after operation between the treatment and control group (P<0.05).3. Bone density change in treatment group and control group in ROI 3 Treatment group:compared with the week after operation, bone density in the year after operation deceased 12.28% (P<0.01);bone density in 3 month after operation decreased 9.43% (P<0.01), bone density in 6 month after operation decreased 11.33%(P<0.01)Control group:compared with the week after operation, bone density in one year after operation deceased 12.30% (P<0.01);bone density in 3 month after operation decreased 9.41% (P<0.01); bone density in 6 month after operation decreased 10.33%(P<0.01).No statistical difference existed in the bone density change in the week after operation between the treatment and control group (P>0.05); No statistical difference existed in the bone density change in the year after operation between the treatment and control group (P>0.05).4. Bone density change in treatment group and control group in ROI 4 Treatment group:compared with the week after operation, bone density in the year after operation deceased 12.85% (P<0.01);bone density in 3 month after operation decreased 5.41% (P<0.01), bone density in 6 month after operation decreased 9.56%(P<0.01).Control group:compared with the week after operation, bone density in one year after operation deceased 14.43% (P<0.01);bone density in 3 month after operation decreased 5.52% (P<0.05); bone density in 6 month after operation decreased 9.46%(P<0.05).No statistical difference existed in the bone density change in the week after operation between the treatment and control group (P>0.05); No statistical difference existed in the bone density change in the year after operation between the treatment and control group (P>0.05).5. Bone density change in treatment group and control group in ROI 5 Treatment group:compared with the week after operation, bone density in the year after operation deceased 21.08% (P<0.01);bone density in 3 month after operation decreased 11.32% (P<0.05), bone density in 6 month after operation decreased 17.51%(P<0.05)Control group:compared with the week after operation, bone density in one year after operation deceased 23.68% (P<0.01);bone density in 3 month after operation decreased 10.92% (P<0.05); bone density in 6 month after operation decreased 17.69%(P<0.01).No statistical difference existed in the bone density change in the week after operation between the treatment and control group (P>0.05); No statistical difference existed in the bone density change in the year after operation between the treatment and control group (P>0.05).6. Bone density change in treatment group and control group in ROI 6 Treatment group:compared with the week after operation, bone density in the year after operation deceased 14.57% (P<0.01);bone density in 3 month after operation decreased 8.29% (P<0.05), bone density in 6 month after operation decreased 13.53%(P<0.01).Control group:compared with the week after operation, bone density in one year after operation deceased 13.17% (P<0.01);bone density in 3 month after operation decreased 7.92% (P<0.05); bone density in 6 month after operation decreased 11.63%(P<0.01)No statistical difference existed in the bone density change in the week after operation between the treatment and control group (P>0.05); No statistical difference existed in the bone density change in the year after operation between the treatment and control group (P>0.05).7. Bone density change in treatment group and control group in ROI 7Treatment group:compared with the week after operation, bone density in the year after operation deceased 11.25% (P<0.01);bone density in 3 month after operation decreased 5.90% (P<0.01), bone density in 6 month after operation decreased 9.39%(P<0.01)Control group:compared with the week after operation, bone density in one year after operation deceased 11.24% (P<0.01);bone density in 3 month after operation decreased 5.85% (P<0.05); bone density in 6 month after operation decreased 9.37%(P<0.01)No statistical difference existed in the bone density change in the week after operation between the treatment and control group (P>0.05); No statistical difference existed in the bone density change in the year after operation between the treatment and control group (P>0.05). 8. Femur and tibia bone density change in the three month, six month and twelve month after operationThree month after operation:in treatment group, femur distal periprosthetic bone density decreased by 11.05%% 10.31%% 9.43%、.41%, with an average of 9.05%, in control group, femur distal periprosthetic bone density decreased by 11.01%、9.96%、9.41%、5.52%, with an average of 8.98%; in treatment group, tibia distal periprosthetic bone density decreased by 11.32%、8.29%、 5.90%, with an average of 8.50%, in control group, tibia distal periprosthetic bone density decreased by 10.92%、7.92%、5.85%, with an average of 8.23%. Six month after operation:in treatment group, femur distal periprosthetic bone density decreased by 18.64%、13.66%、11.33%、9.56%, with an average of 12.85%, in control group, femur distal periprosthetic bone density decreased by 23.74%、18.33%、10.33%、9.46%, with an average of 15.47%; in treatment group, tibia distal periprosthetic bone density decreased by 17.51%、13.53%、9.39%, with an average of 13.48%, in control group, tibia distal periprosthetic bone density decreased by 17.69%、11.63%、9.37%, with an average of 12.90%.Twelve month after operation:in treatment group, femur distal periprosthetic bone density decreased by 20.32%、19.30%、12.28%、12.85%, with an average of 16.19%, in control group, femur distal periprosthetic bone density decreased by 26.26%、25.18%、12.30%、14.43%, with an average of 19.54%; in treatment group, tibia distal periprosthetic bone density decreased by 21.08%、14.57%、11.25%, with an average of 15.63%, in control group, tibia distal periprosthetic bone density decreased by 23.68%、13.17%、11.24%, with an average of 16.03%.二、Clinical Experimental Study( 一) OPG results:OPG results between treatment and control group were equal in the three month before and after operation (P>0.05), the intra-group comparison between two groups showed OPG results was higher after operation than before operation (P<0.01); OPG results in control group in the six month after operation was higher than before operation (P<0.05), OPG results in the six month after was lower than those in the three month after operation (P<0.05); in control group, OPG results in the six month after was equal to those in the three month after operation (P>0.05)(二) RANKL results:RANKL results between treatment and control group were equal in the three month before and after operation (P>0.05), the intra-group comparison between two groups showed RANKL results was higher after operation than before operation (P< 0.05); RANKL results in control group and treatment group were equal in the three month after operation (P<0.05), RANKL results in the control group were higher than those of treatment group in the six month after operation (P<0.05)(三) OPG/RANKL ratio:the ratio between two groups was equal before operation (P>0.05); the ratio between two groups was equal after the three month after operation (P>0.05); the ratio in treatment group was higher than that of control group in the six month after operation (P<0.05).三、 Animal Experimental StudyObservation and area calculation of lacuna skull:Structure of cranium in sham-operation group was normal, no obvious osteolysis or osteoclasia was found. The area of lacuna skull in model was equal to low-dosage and middle-dosage group (P>0.05), the area in high-dosage was the smallest (p<0.01).PCR display:OPG:compared with sham-operation group, mRNA expression in each group decreased (P<0.05); mRNA expression in model group was equal to that of low-dosage group (P>0.05); mRNA expression in high-dosage group was equal to that of middle-dosage group (P>0.05); mRNA expression in high-dosage group was increased compared with low-dosage group (P<0.05).RANKL:compared with sham-operation group, mRNA expression in each group decreased (P<0.05); mRNA expression in model group was equal to that of low-dosage and middle-dosage group (P>0.05); mRNA expression in high-dosage group was lower than that of in middle-dosage group (P<0.05) Western-blot results:OPG:compared with sham-operation group, mRNA expression in each group decreased (P<0.05); mRNA expression in high-dosage group was equal to that of middle-dosage group (P>0.05); mRNA expression in middle-dosage group was higher than that of low-dosage group (P<0.05).RANKL:compared with sham-operation group, mRNA expression in each group increased (P<0.05); mRNA expression in model group was equal to that of low dosage and middle-dosage group (P>0.05); mRNA expression in high-dosage group was lower than that of middle-dosage group (P<0.05).Conclusions:1. Bushen huoxue formula can significantly improve boss density decline and bone mass loss in ROI 1 and ROI2 region in treatment group which indicates, to some extent, the formula could decrease the bone mass loss and could improve the periprosthetical bone absorption after artificial knee joint replacement, although it could not prevent the periprosthetical bone mass loss after surgery.2. Bushen huoxue formula could improve postoperative functions and symptoms after artificial knee joint replacement.3. Bushen huoxue formula could improve symptoms caused by deficiency of liver and kidney, stagnation of tendon and vessel.4. Bushen huoxue formula could increase plasma OPG concentration and OPG/RANKL value.5. In mouse model, high dosage of Bushen huoxue formula could significantly increase mouse skull OPG protein expression and mRNA expression level, could inhibit RANKL protein expression and mRNA expression level, and could increase OPG/RANKL ratio and inhibit osteolysis. |
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