This paper is to show a way of acqusition of the variable region gene of rabbit osteoprotegerin (OPG) and to analyse series. Total RNA was extracted from rabbit tibia, transcripted reversely into cDNA with random primers. The variable region of the OPG gene ampliflied using 5'RACE. Sequencing was confirmed by agarose gel electrophoresis and sequencing analysis. Full length of OPG gene was 1540bp that encoding 400 amino acids. It shared 89% identity with human OPG in whole amino acid sequence and about 85% with rattus norvegicus and other mammal. The OPG sequence of rabbit was obtained by 5'RACE, which could provide a good basis for OPG functional study.
Animals ; Base Sequence ; Molecular Sequence Data ; Osteoprotegerin ; genetics ; Rabbits ; Sequence Homology, Amino Acid

OBJECTIVETo observe the expression of receptor activator of NF-κB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG) during unloading period of dental implants.

METHODSAn animal model of dental implants was established in Beagle dogs. Bone remodeling was tested at 3, 7, 15, 30, 60 and 90 days after the placement of implants. RANKL and OPG mRNA expression were quantified by real-time PCR. Then mandibular bones were resected and some sections were observed.

RESULTSThe most prominent period of bone remodeling occurred at 7 day after the placement of implants (OPG/RANKL mRNA, 2.15 ± 0.1). The expression of RANKL and OPG increased in a time-dependent manner in both soft and hard tissue. After 7 days they gradually decreased.

CONCLUSIONSBoth OPG and RANKL were expressed in peri-implant tissues, and the changing tendency of RANKL and OPGmRNA was consistent with the change of bone remodeling. The active stage for bone remodelling in peri-implant tissues during unloading period is about 7 days after implantation.

Animals ; Bone Remodeling ; genetics ; Dental Implantation ; Dogs ; Male ; Osteoprotegerin ; genetics ; metabolism ; RANK Ligand ; genetics ; metabolism ; RNA, Messenger ; metabolism

OBJECTIVE: To explore the polymorphisms of T149C and T950C gene in osteoprotectin (OPG) promoter sites and the levels of serum OPG and soluble nuclear factor-ΚB receptor activator ligand (sRANKL) and the incidence of coronary heart disease (CHD). METHODS: 528 patients in Tianjin suspected of CHD and underwent coronary angiography (CAG) who admitted to the department of cardiology of Tianjin Chest Hospital from April 2017 to December 2018 were enrolled. According to the CAG results, they were divided into two groups: CHD group (n = 302) and non-CHD group (n = 226). The gender, age, history of hypertension, family history of CHD, diabetes, levels of blood lipid parameters in serum and other clinical data of patients were recorded. The levels of serum OPG and sRANKL were measured by enzyme-linked immunosorbent assay (ELISA). T149C and T950C gene polymorphisms were analyzed by polymerase chain reaction-restriction endonuclease fragment length polymorphism (PCR-RFLP) methods. Hardy-Weinberg genetic balance test was performed for alleles. Binomial classification multivariate non-conditional Logistic regression method was used to analyze the relationship between T149C and T950C gene polymorphisms, serum levels of OPG and sRANKL and CHD. RESULTS: All patients were enrolled in the final analysis. The serum level of OPG in CHD group was significantly higher than that in non-CHD group (μg/L: 1.76±0.49 vs. 1.47±0.29, P < 0.01), the serum level of sRANKL was significantly lower than that in non-CHD group (ng/L: 342.14±121.38 vs. 376.63±108.66, P < 0.05). Logistic regression analysis showed that after adjusting for age, gender, blood lipid parameters, diabetes and other factors, the increase in serum OPG level was an independent risk factor for CHD [odds ratio (OR) = 1.995, 95% confidence interval (95%CI) = 1.935-2.066, P = 0.012]. PCR-RFLP results showed that TT, TC and CC genotypes were found in T149C and T950C of OPG promoter. According to Hardy-Weinberg equilibrium test, the polymorphisms of OPG T149C and T950C accorded with Hardy-Weinberg law, achieving genetic balance with representative of the population. The frequencies of TT, TC, CC and alleles T and C in T149C genotypes of non-CHD group were 53.5%, 42.9%, 3.6%, 75.0% and 25.0%, respectively, and they were 43.1%, 50.3%, 6.6%, 68.2% and 31.8%, respectively in CHD group. There were statistically significant differences in genotype and allele frequencies between the two groups (all P < 0.05). It was shown by Logistic regression analysis that the risk of CHD in TC+CC genotype of T149C was 1.86 of TT genotype (OR = 1.86, 95%CI = 1.24-2.78, P = 0.003). It was suggested that C allele might be a susceptible gene for CHD. In non-CHD group, the frequencies of TT, TC, CC, and alleles T and C in T950C genotypes were 39.8%, 46.5%, 13.7%, 63.1% and 36.9%, respectively. They were 39.4%, 43.4%, 17.2%, 61.1% and 38.9%, respectively in CHD group. There were no significant differences in genotype and allele frequencies between the two groups (all P > 0.05). Logistic regression analysis showed that TC+CC genotype of T950C was not related with CHD. CONCLUSIONS The increased level of serum OPG was closely related with CHD and could be used as a risk factor for CHD. The cases carried OPG T149C TC+CC genotype might have the risk suffering CHD. C allele is might be a susceptible gene.
China/epidemiology* ; Coronary Disease/epidemiology* ; Female ; Humans ; Male ; Osteoprotegerin/genetics* ; Polymorphism, Genetic ; Promoter Regions, Genetic/genetics* ; Risk Factors

OBJECTIVETo investigate the influence of surface modification of titanium on OPG/RANKL mRNA expression in human osteoblast-like cells.

METHODSMG-63 osteoblast-like cells were seeded on the titanium plates with surface polishing and with surface modification by sandblasting plus acid-base treatment, with the cells on glass slides as the control. On days 2, 4, 6, and 8 following cell seeding, the cells were harvested for examination of OPG/RANKL mRNA expression using RT-PCR and real-time PCR.

RESULTSThe expression of OPG/RANKL mRNA was sensitive to the surface microphotography. Compared with the other groups, the cells on the titanium plates with sandblasting plus acid-base treatment, which resulted in a porous micro-structure and high roughness, showed significantly up-regulated expression of OPG mRNA. OPG mRNA expression also showed a time-dependent up-regulation, and was the highest on day 8. The expression of the RANKL mRNA in cells on both of the titanium plates was higher than that in the control cells. The peak level of RANKL mRNA expression occurred on day 6 followed by a gradual decrease.

CONCLUSIONA rough and porous surface of the culture plates and prolonged culture time can synergistically up-regulate the ratio of OPG/RANKL mRNA.

Cell Culture Techniques ; Cell Line ; Humans ; Osteoblasts ; cytology ; metabolism ; Osteoprotegerin ; genetics ; metabolism ; Porosity ; RANK Ligand ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Surface Properties ; Tissue Scaffolds ; Titanium ; chemistry ; pharmacology

OBJECTIVETo study the expression profiles of osteoprotegerin (OPG) and receptor activator of nuclear factor-KB ligand (RANKL) in the distraction region and to investigate the mechanism of bone remodelling during mandibular distraction osteogenesis.

METHODSOsteotomies were performed and external distractors were installed on the mandibles of 42 adult male SD rats. After a 5-day latency period, the distractors were activated at a rate of 0.4 mm/day for 6 days, followed by a 4-week consolidation period. Radiographs were taken, and specimens were harvested at the end of the latency period, when distraction was completed, and at of 1, 2, 3 and 4 weeks of the consolidation period. Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the activated osteoclasts. Temporospatial expression of OPG and RANKL was investigated by using immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). Semi-quantitative analysis was used to characterize OPG,RANKL and RANK/OPG ratio.

RESULTSIn all time points, OPG and RANKL were co-localized in bone marrow lining cells, osteoblasts and newly embedded osteocytes. OPG mRNA expression increased to a peak level when distraction was completed and maintained the level until the end of 2nd week of the consolidation period. RANKL mRNA expression increased steadily until the end of 1 st week of the consolidation period and maintained a peak level until the end of 3rd week, with a slight decrease at the end of 2nd week. The RANKL/OPG ratio increased continuously and reached its highest level at the end of 3rd and 4th week of the consolidation period. TRAP positive osteoclasts were mainly detected at 2, 3 and 4 weeks of the consolidation period in bone marrow cavities and bone surfaces.

CONCLUSIONSThe temporospatial expression patterns of osteoprotegerin and RANKL suggest that osteoblasts and the lineage cell network orchestrates bone remodelling during distraction. Osteogenesis and the most activated bone resorption takes place during 3rd and 4th week of the consolidation phase.

Animals ; Bone Remodeling ; Male ; Mandible ; metabolism ; surgery ; Osteogenesis, Distraction ; Osteoprotegerin ; genetics ; metabolism ; RANK Ligand ; genetics ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo evaluate the possible signal transduction mechanism of the mechanical stress induced by the distraction procedure in osteocytes.

METHODSAn animal model of mandibular distraction osteogenesis in rabbits was established. The expressions of c-fos, OPG and OPGL were detected by ultrasensitive S-P immunohistochemical method.

RESULTAt 4 and 8 days after distraction, distraction zone showed strong positive staining of c-fos, which were apparently higher than that in distraction zone of 2, 4 and 6 weeks after consolidation. At 4 and 8 days after distraction and 2 weeks after consolidation, the expression of OPG was strong, and then wore off gradually at 4 and 6 weeks after consolidation. Weak signals of OPGL could be detected at 6 weeks after consolidation only.

CONCLUSIONc-fos, OPG and OPGL are important regulators in distraction osteogenesis. c-fos is interrelated with the mechanical stress induced by the distraction procedure closely, OPG promotes new bone formation, while OPGL plays a more active role in bone remodeling.

Animals ; Mandible ; cytology ; metabolism ; Osteocytes ; metabolism ; Osteogenesis, Distraction ; Osteoprotegerin ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-fos ; biosynthesis ; genetics ; RANK Ligand ; biosynthesis ; genetics ; Rabbits ; Random Allocation

OBJECTIVEWe investigated the effects of intermittent negative pressure on osteogenesis in human bone marrow-derived stroma cells (BMSCs) in vitro.

METHODSBMSCs were isolated from adult marrow donated by a hip osteoarthritis patient with prosthetic replacement and cultured in vitro. The third passage cells were divided into negative pressure treatment group and control group. The treatment group was induced by negative pressure intermittently (pressure: 50 kPa, 30 min/times, and twice daily). The control was cultured in conventional condition. The osteogenesis of BMSCs was examined by phase-contrast microscopy, the determination of alkaline phosphatase (ALP) activities, and the immunohistochemistry of collagen type I. The mRNA expressions of osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL) in BMSCs were analyzed by real-time polymerase chain reaction (PCR).

RESULTSBMSCs showed a typical appearance of osteoblast after 2 weeks of induction by intermittent negative pressure, the activity of ALP increased significantly, and the expression of collagen type I was positive. In the treatment group, the mRNA expression of OPG increased significantly (P<0.05) and the mRNA expression of OPGL decreased significantly (P<0.05) after 2 weeks, compared with the control.

CONCLUSIONIntermittent negative pressure could promote osteogenesis in human BMSCs in vitro.

Bone Marrow Cells ; cytology ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Humans ; Immunohistochemistry ; Osteogenesis ; Osteoprotegerin ; genetics ; Pressure ; RANK Ligand ; genetics ; RNA, Messenger ; genetics ; Stromal Cells ; cytology

OBJECTIVETo evaluate the effects of sonicated extracts of Porphyromonas gingivalis (Pg) on receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) expression in human periodontal ligament cells (HPDLC) and the effect of Pg on bone resorption in periodontitis.

METHODSHPDLC were exposed to 25, 50 mg/L sonicated extracts of Pg for 6 h, HPDLC without treatment served as control. The expression of RANKL-OPG mRNA and protein were examined by real time polymerase chain reaction and Western blotting. OPG protein in the supernatant was examined by enzyme linked immunosorbent assay (ELISA). The data were statistically analyzed by SPSS 13.0 and one-way analysis of variance (ANOVA).

RESULTSWhen HPDLC were exposed to sonicated extracts of Pg, the expression of RANKL mRNA and protein in 25 mg/L and 50 mg/L groups were higher than that of control group (P < 0.05), the expression of OPG mRNA in 50 mg/L group (0.087 ± 0.021) was lower than that of control group (0.240 ± 0.019) (P < 0.05), and OPG protein in 25 mg/L and 50 mg/L groups (0.813 ± 0.007, 0.398 ± 0.009) was lower than that of control group (1.131 ± 0.005) (P < 0.01). OPG protein expression in the supernatant was not significantly different between experimental group and control group.

CONCLUSIONSSonicated extracts of Pg exposed to HPDLC can up-regulate RANKL expression, down-regulate OPG expression and influence bone metabolism.

Adult ; Cells, Cultured ; Humans ; Osteoprotegerin ; genetics ; metabolism ; Periodontal Ligament ; cytology ; metabolism ; Porphyromonas gingivalis ; pathogenicity ; RANK Ligand ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Sonication ; Young Adult

OBJECTIVETo investigate effects of intermittent negative pressure on osteogenesis in human bone marrow-derived stroma cells (BMSCs) cultured in vitro.

METHODSThe third passage cells were divided into negative pressure treatment group and control group. The cells in the treatment group were induced by negative pressure intermittently (pressure: 17 kPa, 30 min per time, and four times of each day). The cells in the control group were cultured in conventional condition. The osteogenesis of BMSCs was examined by phase-contrast microscopy. The alkaline phosphatase (ALP) activities were determined. The expression of collagen type I was detected by immunohistochemistry method. The mRNA expressions of osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL) in BMSCs were analyzed by real-time polymerase chain reaction (PCR).

RESULTSBMSCs showed a typical appearance of osteoblast after 2 weeks of induction by intermittent negative pressure. The activity of ALP increased significantly, and the expression of collagen type I was positive. In the treatment group, the mRNA expression of OPG increased significantly (P < 0.05) and the mRNA expression of OPGL decreased significantly (P < 0.05) after 2 weeks, compared with the control. However, 3 days after the exposure to 2-week negative pressure, these were no significantly different from that of the control group (P > 0.05).

CONCLUSIONIntermittent negative pressure could promote osteogenesis in BMSCs in vitro.

Bone Marrow Cells ; physiology ; Cell Culture Techniques ; Collagen Type I ; analysis ; Humans ; Osteogenesis ; Osteoprotegerin ; genetics ; Pressure ; RANK Ligand ; genetics ; RNA, Messenger ; analysis ; Stromal Cells ; physiology

Osteoclasts are multinucleated giant cell, which derived from mononuclear myeloid hematopoietic stem cells with the function of bone absorption. Osteoclasts plays a key role in bone metabolism, therefore the body is very strict to regulation of osteoclastogenesis. Mobilization and differentiation of osteoclast maturation is a complex and sophisticated multi-level regulatory processes. In the relevant regulatory mechanisms, OPG/RANKL/RANK system plays a pivotal role in the process of osteoclast differentiation and maturation. Recent studies revealed that immune cells and osteoclasts were closely connect with each other in the field of bone metabolism, also provide a new therapeutic target for the treatment of bone diseases. The apoptosis of osteoclasts in bone metabolism have been payed more attention,while its mechanism is still not clear, which need further research.
Animals ; Cell Differentiation ; Gene Expression Regulation ; Humans ; Osteoclasts ; cytology ; metabolism ; Osteoprotegerin ; genetics ; metabolism ; RANK Ligand ; metabolism ; Receptor Activator of Nuclear Factor-kappa B ; genetics ; metabolism