Objective To investigate the association between the genetic polymorphisms of osteoprotegerin gene and bone mineral density ( BMD) in elderly patients with chronic obstructive pulmonary disease ( COPD) .Methods 178 elderly COPD patients admitted in respiratory department between January 2008 and December 2009 were recruited as a COPD group. 195 elderly healthy subjects without COPD were recruited as a control group. The subjects were all chosen from the Han population in Lanzhou city, Gansu province. Pulmonary function ( FEV1 /FVC, FEV1% pred) , body mass index ( BMI) , serum calcium ( Ca) , serum phosphate ( P) , and alkaline phosphatase ( ALP) were determined in all subjects. The OPG gene polymorphisms were analyzed by polymerase chain reaction and restriction fragment length polymorphism ( PCR-RFLP) . BMD was examined by dual-energy X-ray absorptiometry. Results In the COPD group, the distribution frequency of AAGG, GATA, and GGTT in OPG HTT gene-linked polymorphic region G209A and T245G were 2.5%, 27.2% , and 72.3% , respectively, which in the control group were 2.2% , 26.9% , and 70.9%, respectively. The genotype distribution difference of two groups had no statistical significance ( P gt; 0.05) . There were also no statistical differences in BMI, serum Ca, serum P, serum ALP or BMD between different genotype subgroups in two groups ( P gt;0.05) . In the COPD group, the genotype distribution had no statistical significance between different BMD subgroups( P gt; 0.05) . Conclusion In the elderly patients with COPD from Han population at Lanzhou city, OPG HTT gene-linked polymorphic region and T245G gene polymorphism have no significant correlation with reduced lung function, reduced BMD and bone metabolism which are not likely to be susceptibility loci for osteoporosis in COPD patients.
Objective To investigate the effect of glucocorticoid on the expression levels of osteoprotegerin (OPG)/receptor activator of nuclear factor kappa B ligand (RANKL)-matrix metalloproteinases (MMP)/tissue inhibitor of matrix metalloproteinase (TIMP) system in bone tissues of femoral head of rats, and to discuss its interrelated action mechanism in glucocorticoid-induced avascular necrosis of femoral head (ANFH). Methods Forty adult Sprague Dawley rats, weighing 250-300 g, half males and half females, were randomly divided into 4 groups: high dose glucocorticoid group (HD, n=10), medium dose glucocorticoid group (MD, n=10), low dose glucocorticoid group (LD, n=10), and control group (n=10). The rats in HD group, MD group, and LD group were intramuscularly injected with 25.0, 12.5, and 7.0 mg/kg of prednisolone respectively, and the rats in the control group were injected with physiological saline. After 4 weeks intervention, the osteonecrosis of left femoral heads was observed by HE staining, total RNA was extracted from the right femoral head bone tissue and the mRNA expression levels of OPG, RANKL, MMP-2, MMP-9, TIMP-1, and TIMP-2 were detected by RT-PCR. Results After injection of prednisolone, 4 rats of HD group and 1 rat of MD group died of systemic failure caused by the decreased food and weight culminating in cachexia. HE staining showed that the integrity of bone trabecula and osteon was destroyed at different levels, discontinuous bone chips formed, and osteocytes were replaced by granulation tissue in some lacunae in HD, MD, and LD groups; the integrated osteon was observed, the lamellar structure formed concentric circles around the blood vessel and osteocytes were seen in the lacunae in the control group. The necrosis rates of femoral head were 83.3% (5/6), 66.7% (6/9), 30.0% (3/10), and 0 (0/10) in HD, MD, LD, and control groups. The results of RT-PCR showed: the mRNA expression levels of the OPG, TIMP-1, TIMP-2 in HD, MD, and LD groups were lower than those in the control group, showing significant differences (P lt; 0.05) and there was negative correlation with the hormone dosage. The difference in OPG expression was significant between the hormone groups (P lt; 0.05); the differences in the TIMP-1 and TIMP-2 expressions were not significant between the LD group and MD group (P gt; 0.05), but there were significant differences when compared with HD group (P lt; 0.05). The RANKL, MMP-2, and MMP-9 mRNA expression levels in HD, MD, and LD groups were higher than those in the control group and there was a positive correlation with the hormone dosage, showing significant differences when compared MD and HD groups with control group (P lt; 0.05); there was no significant difference in RANKL expression between HD group and MD group (P gt; 0.05), but there was significant difference when compared HD and MD groups with LD group (P gt; 0.05); no significant difference was observed in the MMP-2 and MMP-9 expression between MD group and LD group (P gt; 0.05), but the differences were significant when compared with HD group (P lt; 0.05). Conclusion Glucocorticoid-induced ANFH may be related to the expression levels of OPG/RANKL-MMP/TIMP mRNA regulated by glucocorticoid.
Objective To evaluate the effects and the molecular mechanism of Liuwei dihuang pills in preventing steroid-induced osteonecrosis of the femoral head (ONFH) so as to provide an expremental basis for preventing ONFH cl inically. Methods Thirty-six adult Kunming mice (weighing 40-50 g, 46 g on average) were randomly divided into three groups (n=12): group A (control group), group B (model group) and group C (prevention group). In groups B and C, ONFHmice models were produced by intraperitoneal injection of horse serum at first (10 mL/kg) and a second injection of horse serum intraperitoneally (5 mL/kg) and prednisolone intramuscularly [45 mL/(kg•day), for 5 days] 2 weeks later. At the same time, the mice in group C were given Liuwei dihuang pills intragastrically [2 g/(kg•day)] and were given normal sal ine [10 mL/(kg•day)] in group B. In group A, mice were given normal sal ine intramuscularly and intragastrically as controls. The animals were sacrificed 2, 4, and 8 weeks after first treatment with prednisone, and femoral heads and l ivers were harvested to do histopathology analysis and apoptosis assay. Results Other mice survived throughout the experiment period except two death at 7 and 11 days after second injection of horse serum intraperitoneally in group B and one death at 24 hours after second injection of horse serum intraperitoneally in group C. The appearance and shape of the femoral head and the surface of cartilages were all normal. The histological observation showed: normal structures of l iver and femoral head were seen in group A at each time point; swell ing l iver cells with small fat vacuole, unclear structure of hepatic cords and narrower sinus hepaticus were seen, the bone trabeculae of femoral head was thin, sparse and collapsed in some regions and the changes became more obvious with time in group B; group C had similar results to group A. The percentage of empty osteocyte lacunae was significantly higher in group B than in groups A and C (P lt; 0.01). The osteoprotegrin expression significantly decreased and the osteoprotegrin l igand expressionsignificantly increased in group B when compared with groups A and C (P lt; 0.01). Apoptosis analysis showed that the apoptosis index in group B was significantly higher than that in groups A and C (P lt; 0.01). Conclusion Liuwei dihuang pills can prevent steroid-induced ONFH by improving l ipid metabol ism, releiving bone lesion, and protecting against cell death.
Objective Lots of metal ions accumulation and over-expression of receptor activator of NF-κB l igand (RANKL) around the prosthesis could be found in revision of total hip arthroplasty. To investigate the relationship between metal ions and aseptic loosening by observing the effects of Co2+ and Cr3+ ions on the expression of RANKL and osteoprotegerin(OPG) from osteoblast. Methods Osteoblasts were cultured in vitro at the density of 1 × 105 cells/mL, and were divided into 2 groups according to different culture solutions. In control group, osteoblasts were cultured with normal medium without CoCl2 and CrCl3. In experimental group, osteoblasts were cultured with the medium including CoCl2 (10 mg/ L) and CrCl3 (150 mg/L) solutions. The RT-PCR and ELISA methods were appl ied to detect the mRNA expression of RANKL and OPG and protein level at 24 and 48 hours after co-cultured, respectively. Results RT-PCR revealed that the mRNA expression of RANKL and OPG could be found in two groups at 24 and 48 hours after co-cultured, the expression was higher in the experimental group than in control group, especially the expression of RANKL, showing significant difference (P lt; 0.05). At 24 and 48 hours after co- cultured, the ratios of RANKL mRNA to OPG mRNA in the experimental group were 0.860 and 1.232, respectively, which were significantly higher than those in the control group (0.695 and 0.688,P lt; 0.05). ELISA revealed that the protein level of RANKL and OPG in experimental group were significantly higher than those in the control group (P lt; 0.05). Conclusion Co2+ and Cr3+ can stimulate the mRNA expressions of RANKL, OPG and secretion of those protein from osteoblasts, especially increase of the RANKL, which promotes the formation and activation of osteoblasts and the generation of aseptic loosening.
Objective To investigate the effects of intermittent negative pressure on the mRNA expression of osteoprotegerin (OPG) and osteoprotegerin l igand (OPGL) in human BMSCs cultured in vitro. Methods BMSCs were isolated from adult marrow donated by 2 hip osteoarthritis patients with prosthetic replacement in January 2008 and cultured in vitro. The third passage cells were divided into experimental group and control group. The experimental group was induced by negative pressure intermittently for 2 weeks (pressure: 50 kPa, 30 minutes each time, twice per day) and the control groupwas routinely cultured. After 2 weeks of culture, cell morphology was observed by inverted phase contrast microscope, and the mRNA expressions of OPG and OPGL in BMSCs were analyzed by real-time PCR. Results The cell prol iferation speed of the experimental group was slower than that of the control group. The cell morph changed from shuttle to megagon with some prominences in experimental group and the cell morph kept shuttle in the control. The mRNA expression of OPG in experimental group increased significantly (P lt; 0.01) and the mRNA expression of OPGL in experimental group decreased significantly compared with control group (P lt; 0.01) 2 weeks later. Conclusion Intermittent negative pressure is capable of promoting the expression of OPG, while inhibiting the expression of OPGL in human BMSCs.
OBJECTIVE: To construct a co-expressing vector of human bone morphogenetic protein 2 (BMP-2) and osteoprotegerin (OPG) and to determine the expression of BMP-2 and OPG in myoblast C2C12. METHODS: Using the isolated total RNA from osteosacoma cell line MG63 as a template, the cDNA encoding region of human OPG was amplified by reverse transcription-polymerase chain reaction (RT-PCT) method and cloned into sites EcoR 1 and BamH I of mammalian expressing vector pIRES2-EGFP, and the cDNA encoding region of human BMP-2 was cloned into endonucleases site BstX I. Then the recombinant plasmid pIRES2-BMP-2-OPG was transformed into C2C12 cell line, the expression of OPG and BMP-2 were determined by Western blot assay. RESULTS: The sequence of OPG cDNA obtained was the same as that reported, recombinant plasmid pIRES2-BMP-2-OPG was constructed successfully. Human OPG and BMP-2 co-expression cell line C2C12 was selected and confirmed by Western blot analysis. CONCLUSION: The co-expressing vector of OPG and BMP-2 is constructed and can expressed stably in myoblast C2C12. The co-expression of human OPG and BMP-2 may be logical approach for treatment of osteoporosis and bone metastasis.
ObjectiveTo investigate the relationship among bone density, osteoprotegerin (OPG) and vascular calcification (VC) in maintenance hemodialysis (MHD) patients. MethodsOne hundred MHD patients were collected from our department between May 2010 and December 2012. The VC was detected by plain radiographs. Bone mineral density was measured with dual-energy X-ray absorptionmeter. The level of serum OPG was measured by enzymelinked immunosorbent assay. Other clinically related indicators were also detected. The related parameters were examined statistically. ResultsThe incidence of VC in MHD patients was 74% (74/100), and the OPG level significantly increased with the degree of vascular calcification (P<0.05). The proportion of patients with normal bone volume was 40%, and with abnormal bone volume was 60%. Compared with patients with normal bone volume, the patients with abnormal bone volume had higher serum OPG level (P<0.05). The patients with no VC had a lower incidence of abnormal bone volume than patients with VC (P<0.05). Multiple linear regression analysis revealed that vascular calcification score, OPG level and age were independent factors for bone mineral density. Dialysis time, OPG level, serum albumin level and bone mineral density were independent factors for vascular calcification score. ConclusionThe MHD patients with vascular calcification are often associated with osteoporosis at the same time. OPG plays an important role in the relationship between vascular calcification and osteoporosis.
ObjectiveTo investigate the effects of different concentrations of osteoprotegerin (OPG) combined with deproteinized bone (DPB) on the bone tunnel after the anterior cruciate ligament (ACL) reconstruction. MethodsThe femoral epiphyseal side was harvested from newborn calf, and allogenic DPB were prepared by hydrogen peroxide-chloroform/methanol method. Then, DPB were immersed in 3 concentrations levels of OPG (30, 60, 100 μg/mL) and 3 concentration ratios (30%, 60%, 100%) of the gel complex were prepared. Sixty healthy New Zealand white rabbits, male or female, weighing (2.7±0.4) kg, were divided randomly into 4 groups (n=15):control group (group A), 30% (group B), 60% (group C), and 100% (group D) OPG/DPB gel complex. The ACL reconstruction models were established by autologous Achilles tendon. Different ratios of OPG/DPB gel complex were implanted in the femoral and tibial bone tunnel of groups B, C, and D, but group A was not treated. The pathology observation (including the percentage of the femoral bone tunnel enlargement) and histological observation were performed and the biomechanical properties were measured at 4, 8, and 12 weeks after operation. ResultsOne rabbit died of infection in groups A and D, 2 rabbits in groups B and C respectively, and were added. General pathology observation showed that the internal orifices of the femoral and tibia tunnels were covered by a little of scar tissue at 4 weeks in all groups. At 8 weeks, white chondroid tissues were observed around the internal orifices of the femoral and tibia tunnels, especially in groups C and D. At 12 weeks, the internal orifices of the femoral and tibia tunnels enlarged in groups A, B, and C, but it was completely closed in group D. At each time point, the rates of the femoral bone tunnel enlargement in groups B, C, and D were significantly lower than that in group A, and group D was significantly lower than groups B and C (P<0.05); group C was significantly lower than group B at 8 weeks, but no significant difference was found at 4 and 12 weeks (P<0.05). Hisological observation showed that fresh fibrous connective tissue was observed in 4 groups at 4 weeks; there was various arrangements of Sharpey fiber in all groups at 8 weeks and the atypical 4-layer structure of bone was seen in group D; at 12 weeks, Sharpey fiber arranged regularly in all groups, with typical 4-layer structure of bone in groups B, C, and D, and an irregular "tidal line" formed, especially in group D. Biomechanics measurement showed that the maximum tensile load in group D was significantly higher than that in groups A and B at 4 weeks (P<0.05), but no significant difference was shown among groups A, B, and C, and between groups C and D (P>0.05); at 8 weeks, it was significantly higher in groups C and group D than group A, and in group D than group B (P<0.05), but there was no significant difference between groups A, C and group B (P>0.05); at 12 weeks, it was significantly higher in groups C and D than groups A and B, and in group D than group C (P<0.05), but difference was not significant between groups A and B (P>0.05). ConclusionDifferent concentrations ratios of OPG/DPB gel complexes have different effects on the bone tunnel after ACL reconstruction. 100% OPG/DPB gel complex has significant effects to prevent the enlargement of bone tunnel and to enhance tendon bone healing.
Objective To investigate the correlation of elderly knee osteoarthritis with bone marrow edema and osteoprotegerin, DKK-1 (dickkopf-1), sclerostin. Methods A total of 100 elderly patients with knee osteoarthritis in Sichuan Province Orthopedic Hospital from September 2017 to December 2018 were selected and divided into bone marrow edema group (50 cases) and non-bone marrow edema group (50 cases). The patients’ basic data, Western Ontario and McMaster University Osteoarthritis Index (WOMAC) scores and Visual Analogue Scale scores were collected. The patients’ serum osteoprotegerin, DKK-1, sclerostin, C-reactive protein, and erythrocyte sedimentation rate were tested, and the differences between the two groups were compared. The correlation of the detection indicators and bone marrow edema and its clinical indicators was explored. Results There was no significant difference in age, gender, course of disease, C-reactive protein and erythrocyte sedimentation rate between the two groups (P>0.05). WOMAC scores (76.1±5.4 vs. 67.5±6.6), Visual Analogue Scale scores (8.4±1.1 vs. 5.5±0.9), proportion of synovitis (84.0% vs. 52.0%), osteoprotegerin [(1.3±1.1) vs. (0.6±0.5) μg/L], DKK-1 [(18.4±16.9) vs. (6.9±6.0) μg/L] and sclerostin [(147.3±119.4) vs. (99.7±70.7) pg/mL] in the bone marrow edema group were higher than those in the non-bone marrow edema group (P<0.05). There was no statistically significant correlation of the bone marrow edema volume score and degree score and serum osteoprotegerin of patients in the bone marrow edema group (P>0.05). The bone marrow edema volume score and degree score of patients in the bone marrow edema group were positively correlated with serum DKK-1 (volume score rs=0.464, P=0.001; degree score rs=0.379, P=0.007) and sclerostin (volume score rs=0.316, P=0.025; degree score rs=0.461, P=0.003). Conclusion In elderly patients with knee osteoarthritis and bone marrow edema, the local bone metabolism indicators of osteoprotegerin, DKK-1 and sclerostin are up-regulated, especially DKK-1 and sclerostin are related to the severity of bone marrow edema.