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

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 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

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

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

OBJECTIVETo investigate the expression of receptor activator of nuclear factor-κB (RANK), its ligand RANKL, and osteoprotegerin, and observe the effects of αD3 on their expressions in male rats at different ages.

METHODSWistar rats at 6 weeks, 6 months, and 24 months (n=15) were examined for mRNA expressions of RANK/RANKL and osteoprotegerin in the left proximal femur using RT-PCR and for their protein expressions in the right femur using immunohistochemistry. RANK/RANKL and osteoprotegerin expressions were also detected in another 15 rats aged 24 months following intragastric administration of 0.05 µg/kg αD3 (3 times a week for 10 weeks).

RESULTSCompared with 6-week-old rats, 6-month- and 24-month-old rats showed a 6.2-fold and 7.3-fold increase of RANKL mRNA expression, respectively (P<0.05), and osteoprotegerin mRNA levels increased slightly with age. αD3 treatment resulted in significantly increased expression of RANK in 24-month-old rats with a lowered RANKL/osteoprotegerin ratio. RANKL and osteoprotegerin were co-localized in the osteoblasts and chondrocytes. αD3 treatment also caused an increased expression of osteoprotegerin mRNA in 24-month-old rats.

CONCLUSIONThe age-related increase of the ratio of RANKL/osteoprotegerin mRNA promotes osteoclast activity and bone turnover. αD3 has favorable effect on osteogenesis and suppress bone absorption in the femur possibly by reducing RANK expression and lowering RANKL/osteoprotegerin ratio.

Age Factors ; Animals ; Chondrocytes ; metabolism ; Femur ; metabolism ; Male ; Osteoblasts ; metabolism ; Osteoprotegerin ; genetics ; metabolism ; RANK Ligand ; genetics ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; Receptor Activator of Nuclear Factor-kappa B ; genetics ; metabolism

OBJECTIVETo explore the mechanism of Shangke Jiegu Tablet (SJT)in repairing the mandibular defect.

METHODSTotally 72 healthy male New Zealand rabbits were randomly divided into the normal control group (n = 24), the model group (n = 24), and the SJT group (n = 24). Then the mandibular defect model was established. Animals in the normal control group and the model group were fed with normal forage, while those in the SJT group were fed with SJT forage. On the day 7, 14, 28, and 56 after model establishment, 6 rabbits were killed in each group. The bone was collected from the mandibular defect. The gene expressions of osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL) were detected by means of RT-PCR. The positive dyeing strength and area of the bone tissue were detected by means of immunohistochemical technique.

RESULTSCompared with the normal control group, the degree of OPGmRNA expression was remarkably up-regulated on day 7 after model establishment (P < 0.05) and the degree of OPGLmRNA expression was remarkably up-regulated on day 14 after model establishment (P < 0.05) in the model group. Compared with the model group, the degree of OPGmRNA expression was remarkably up-regulated (P < 0.05), and the positive dyeing strength and area of bone tissue were stronger and broader on day 14, 28, and 56 after model establishment in the SJT group. The degree of OPGLmRNA expression was remarkably down-regulated (P < 0.05), and the positive dyeing strength and area of bone tissue were weaker and smaller on day 14 after model establishment in the SJT group. The ratio of OPGmRNA/OPGLmRNA was remarkably up-regulated on day 14, 28, and 56 after model establishment (P < 0.05).

CONCLUSIONThe effect mechanism of promoting mandibular defect repairing by SJT may be correlated to regulating the expressions of OPGmRNA and OPGLmRNA.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression ; drug effects ; Ligands ; Male ; Mandibular Injuries ; genetics ; metabolism ; Osteoprotegerin ; metabolism ; RANK Ligand ; metabolism ; Rabbits ; Wound Healing ; drug effects

OBJECTIVETo observe the effect of treadmill exercise on the mRNA expression of osteoprotegerin, RANKL and RUNX2 in bone tissue of ovariectomized rat,and to investigate the mechanism of treadmill exercise for the preventing and treating postmenopausal osteoporosis.

METHODSThirty healthy adult SD rats which average weight was (270 +/- 10) g were randomly divided into 3 groups: sham-operation group, ovariectomized group and treadmill exercise group. Each group had 10 rats. After anesthesia, ovariectomized group and treadmill exercise treated group were operated by bilateral ovarian resection, sham group by sham operation. Sham-operation group and ovarian rats were normal breed. Rats in the exercise group were treated by running on treadmill for animal at 1 week after the operation. Running speed was 18 meters per minutes lasting 45 minutes in one day, and worked six days per week for 11 weeks. All rats were killed after 12 weeks. After decalcification, left femur head was made to paraffin slice and observed by inverted phase contrast microscope for histological examination. Total RNA were extracted from the right femur heads and the mRNA expression of OPG,RANKL and RUNX2 were examined by real time PCR.

RESULTSThe femur heads of ovariectomized group showed thin trabecular bone and less bone cells, meanwhile trabecular bone of exercise group looked thicker in histological examination. The mRNA expression of OPG (0.131 +/- 0.080), RANKL (8.013 +/- 3.550) and RUNX2 (3.245 +/- 5.090) was seen in the ovariectomized group. Meanwhile different results were found as the mRNA expression of OPG (0.566 +/- 0.260), RANKL (5.232 +/- 3.670) and RUNX2 (2.753 +/- 3.680) in the group of treadmill exercise. In compared with ovariectomized group, the mRNA expression of OPG in treadmill exercise increased and the result had statistical significance,whereas mRNA expression of RNAKL and RUNX2 decreased but the results had no statistical significance.

CONCLUSIONThe effect of exercise treating postmenopausal osteoporosis is tightly correlated with the up-regulation of expression of OPG. This provides a new train of thought of postmenopausal osteoporosis treatment for the future study.

Adult ; Animals ; Bone Density ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Exercise Test ; Exercise Therapy ; Female ; Femur ; metabolism ; Humans ; Osteoporosis, Postmenopausal ; genetics ; metabolism ; physiopathology ; therapy ; Osteoprotegerin ; genetics ; metabolism ; Ovariectomy ; RANK Ligand ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley

BACKGROUNDOsteoprotegerin (OPG) and osteoprotegerin ligand (OPGL) play an important role in human bone metabolism. The aim of this research was to detect the expression of OPG and OPGL during human marrow stromal cells (hMSC) differentiation into osteoblasts (OB), and to observe their effect on osteoclasts (OC) formation in vitro to investigate bone metabolism mechanisms.

METHODShMSCs were obtained from human bone marrow specimens using gradient centrifugation method, before being purified and incubated with differentiation medium to develop along the human osteoblasts (hOB) pathway. Morphology observation, biochemical detection and cell staining were performed during hMSC differentiation. OPG and OPGL mRNA levels were detected by reverse transcription-polymerase chain reaction. OPG and OPGL protein expression were determined by Western blotting. We further obtained OC progenitor cells from mice bone marrow and co-cultured with differentiating MSCs. We assessed the effect of OPG and OPGL on OC formation by identifying tartrate resistant acid phosphatase (TRAP) positive multinuclear cells.

RESULTSOptimal hMSC survival and purification were observed, along with stable biochemical indexes. Alkaline phosphatase secretion increased significantly and mineralization nodules appeared in the process of cell differentiation. OPG mRNA and protein level increased significantly, while OPGL mRNA and protein level decreased. Average levels of OPG mRNA and protein were about 2.5-fold higher than the control, while OPGL mRNA and protein levels were reduced by about one-half. In the group co-culturing with undifferentiated MSC or added OPGL, we found TRAP positive and multi- nuclear OC formation. However, OC formation was absent in the group co-culturing with differentiated MSC or added OPG.

CONCLUSIONSDuring hMSC differentiation into hOB, OPG secretion increased rapidly and OPGL production decreased significantly. The OPG/OPGL ratio was also increased, while OC formation was inhibited and bone absorption decreased. Thus, regulation of the OPG/OPGL ratio may be important in controlling MSC differentiation, OB and OC formation in succession involved in bone metabolism.

Adult ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; genetics ; physiology ; Cells, Cultured ; Female ; Humans ; Male ; Middle Aged ; Osteoclasts ; cytology ; metabolism ; Osteoprotegerin ; genetics ; metabolism ; RANK Ligand ; genetics ; metabolism ; Stromal Cells ; cytology ; metabolism