Osteoprotegerin Receptor Activator Of Nuclear factor-kappaB Ligand Research Articles

Biomarkers in External Apical Root Resorption: An Evidence-based Scoping Review in Biofluids.

External apical root resorption (EARR), an unwanted sequela of orthodontic treatment, is difficult to diagnose radiographically. Hence, the current scoping review was planned to generate critical evidence related to biomarkers in oral fluids, i.e. gingival crevicular fluid (GCF), saliva, and blood, of patients showing root resorption, compared to no-resorption or physiologic resorption. A literature search was conducted in major databases along with a manual search of relevant articles in the library, and further search from references of the related articles in March 2021. The initial search was subjected to strict inclusion and exclusion criteria according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Following PRISMA guidelines, 20 studies were included in the final review. The studies included human clinical trials and cross-sectional and prospective studies with/without control groups with no date/language restriction. Various biomarkers identified in EARR included dentinal proteins, enzymes, cytokines, and salivary proteins. Severe resorption had higher dentin sialoprotein (DSP) and resorption protein concentrations as well as lower granulocyte-macrophage colony-stimulating factor (GM-CSF) as compared with mild resorption. Increased DSP and dentin phosphophoryn (DPP) expression was found in physiologic resorption. Compared to controls, resorbed teeth showed a higher receptor activator of nuclear factor kappa B ligand/osteoprotegerin (RANKL/OPG) ratio. In contrast, levels of anti-resorptive mediators (IL-1RA, IL-4) was significantly decreased. Differences in force levels (150 g and 100 g) showed no difference in resorption, but a significant rise in biomarkers (aspartate transaminase [AST] and alkaline phosphatase [ALP]) for 150 g force. Moderate to severe resorption in young patients showed a rise in specific salivary proteins, requiring further validation. Limitations of the studies were heterogeneity in study design, biomarker collection, sample selection, and confounding inflammatory conditions. Various biomarkers in biofluids indicate active resorption, while resorption severity was associated with DSP and GM-CSF in GCF, and a few salivary proteins. However, a robust study design in the future is mandated.

Dark-light cycle disrupts bone metabolism and suppresses joint deterioration in osteoarthritic rats

BackgroundLight alteration affects the internal environment and metabolic homeostasis of the body through circadian rhythm disorders (CRD). CRD is one of the factors that induce and accelerate osteoarthritis (OA). Therefore, the aim of this study was to evaluate the effects of continuous dark-light (DL) cycle on joint inflammation, bone structure, and metabolism in normal and OA Sprague-Dawley (SD) rats.MethodsInterleukin (IL)-1β, IL-6, inducible nitric oxide synthase (iNOS), and tumor necrosis factor (TNF)-α were used to evaluate the systemic inflammation in rats. The pathological changes and inflammatory reactions of the cartilage and synovium of the knee joint in rats were evaluated by Safranin O-fast green and immunological staining. Bone turnover was assessed by histomorphometry and μCT scanning, as well as bone metabolism markers and proteins. The expression changes of clock proteins BMAL1, NR1D1, PER3, and CRY1 in representative tissues were detected by western blotting.ResultsDL cycle significantly inhibited body weight gain in normal and OA rats. The levels of proinflammatory factors in the peripheral blood circulation and degradation enzymes in the cartilage were significantly decreased in OA+DL rats. DL cycle significantly destroyed the structure of subchondral bone in hindlimbs of OA rats and reduced trabecular bone numbers. The decrease of bone mineral density (BMD), percent bone volume with respect to total bone volume (BV/TV), trabecular number (TB.N), osteoclast number, and mineralization could also be found. The ratio of the receptor activator of nuclear factor-kappa B ligand/osteoprotegerin (RANKL/OPG) in the bone marrow of OA rats was markedly increased under DL, along with the activation of the mononuclear/phagocyte system. The expression of representative clock proteins and genes BMAL1, PER3, and CRY1 were markedly changed in the tissues of OA+DL rats.ConclusionsThese results suggested that DL cycle dampened the arthritis and promoted bone resorption and bone mass loss.Graphical abstractDL cycle affects bone turnover by regulating osteoclast production in osteoarthritic rats.

Experimental study on the regulation of subchondral bone plate osteoprotegerin/receptor activator of nuclear factor kappa-B ligand system by berberine to retard the development of osteoarthritis in rabbits

To investigate the effect of intra-articular berberine injection on the structural remodeling of subchondral bone plate and osteoprotegerin/receptor activator of nuclear factor kappa-B ligand(OPG/RANKL) system expression in rabbits with osteoarthritis(OA). Forty 12-month-old male rabbits with an average of(2.73±0.18) kg of body weight, underwent left anterior cruciate ligament transection(ACLT), and were divided into berberine group and placebo groups after operation, 20 rabbits in each group. The berberine group received intra-articular injection of 100 μmol/L berberine 0.3 ml every week for 6 weeks. In placebo group, the same dose of 0.9% sodium chloride injection was injected into the left knee joint cavity every week for 6 weeks. Another 20 12-month-old male rabbits, weighing (2.68±0.18) kg, underwent sham operation on the left knee joint without intra-articular injection intervention (sham operation group). On the last day of the sixth week after operation, three groups of animals were sacrificed to obtain knee joint specimens. The femoral medial condyle samples were obtained for histological evaluation of cartilage and subchondral bone, Mankin scoring system was used to evaluate articular cartilage structure. Image-Pro Plus(IPP) software was used to evaluate subchondral bone plate bone volume(BV), bone volume/total volume(BV/TV), trabecular circumference(TC), mean trabecular thickness (Tb.Th). Real-time quantitative reverse transcription polymerization Enzyme chain reaction(reverse transcription-polymerase chain reaction, RT-PCR) was used to detect the mRNA expression levels of OPG and RANKL in subchondral bone tissue at 6 weeks after operation. The cartilage structure evaluation showed that the surface of cartilage tissue in the sham operation group was smooth and flat, and the safranin coloration was full in the full thickness of the cartilage;the cartilage tissue in the berberine group showed uneven surface layer, and the staining of safranin O was mildly decreased;the surface layer fibrosis was seen in placebo group, Safranin O faded significantly. The Mankin score in the berberine group was lower than that in placebo group(P<0.01), but higher than that in sham operation group(P<0.01). The structural evaluation of subchondral bone plate showed that the trabecular bone in sham-operated group was densely arranged;after berberine intervention, the trabeculae were closely arranged;the subchondral bone trabeculae in placebo group were relatively sparse, and the distance between trabeculae was wider. Subchondral bone plate IPP software evaluation showed that BV, BV/TV, TC, Tb.Th in berberine group were higher than those in placebo group(P<0.01), BV, BV/TV, TC, Tb.Th in berberine group were higher than those in placebo group(P<0.01), while lower than the sham operation group (P<0.01). PCR test results showed that the expression of OPG mRNA in the berberine group was significantly higher than that in placebo group(P<0.01), and OPG mRNA in the berberine group was lower than that in sham operation group (P<0.01). There was no significant difference in mRNA expression of RANKL among three groups(P>0.05);the ratio of OPG/RANKL in berberine group was higher than that in placebo group(P<0.01), but lower than that in sham operation group(P<0.01). Intra-articular injection of berberine can effectively inhibit the resorption of subchondral bone in the early stage of OA and delay the development of the disease. The specific mechanism may be that berberine maintains the balance of OPG/RANKL system by up-regulating the expression of OPG gene in subchondral bone.

The effect of vitamin D deficiency on the RANKL/OPG ratio in rats

The aim of this study was to determine the effect of vitamin D deficiency on the RANKL/OPG ((Receptor Activator of Nuclear Factor Kappa B Ligand/Osteoprotegerin) ratio in the serum blood levels. Sixteen Wistar rats were divided into 2 groups: a control group (C) and an experimental group (E). The group C comprised of rats with average vitamin D levels, while vitamin D deficiency was induced in those of group E. A test period of 21 days was employed wherein two serum blood samples were obtained, at the baseline (day 0) and at day 21. Changes in RANKL andOPG levels were measured using Enzyme-linked immunosorbent assay (ELISA) kits. A statistically significant decrease of serum RANKL concentration and RANKL/OPG ratio on day 21 was seen in the experimental group compared to the control group. The serum RANKL levels and RANKL/OPG ratio in rats, were negatively affected by the deficiency of vitamin D.

Diabetes mellitus accelerates the progression of osteoarthritis in streptozotocin-induced diabetic mice by deteriorating bone microarchitecture, bone mineral composition, and bone strength of subchondral bone.

BackgroundThe purpose of this study was to develop an optimal diabetes-osteoarthritis (DM-OA) mouse model to validate that diabetes aggravates osteoarthritis (OA) and to evaluate the microarchitecture, chemical composition, and biomechanical properties of subchondral bone (SB) as a consequence of the DM-OA-induced damage induced.MethodsMice were randomly divided into three groups: DM-OA group, OA group, and sham group. Blood glucose levels, body weight, and food intake of all animals were recorded. Serum calcium (Ca) and osteocalcin (OCN) levels were compared in the three groups. The messenger ribonucleic acid (mRNA) and protein expression of key regulators for bone metabolism were detected. A semi-quantitative grading system [Osteoarthritis Research Society International (OARSI)] was used to evaluate cartilage and SB degeneration. Microspectroscopy, microindentations, micro-computed tomography (CT) imaging, and fracture load of compression testing were also used to evaluate trabecular SB properties.ResultsGlycemic monitoring and pancreas pathological results indicated stable high blood glucose and massive destruction of pancreas and islet cells in the DM-OA group. Serum levels of bone specific alkaline phosphatase (ALP-B) and tartrate-resistant acid phosphatase 5b (TRACP-5b) in the DM-group were higher than those of the other two groups while levels of serum Ca and OCN were lower. Meanwhile, the protein and mRNA expression of osteoblast-specific biomarkers [osteoprotegerin/receptor activator of nuclear factor kappa-B ligand (OPG/RANKL) ratio, collagen type I (COL-I), Runt-related transcription factor 2 (RUNX-2), OCN] were suppressed, and osteoclast-specific biomarkers [sclerostin (SOST)] was elevated in the DM-OA group. The mineral-to-collagen ratio, microindentation elastic modulus, hardness, micro-architectural parameters, bone mineral density, and fracture load of SB trabecular bone of the DM-OA group joint were lower than those of the other two groups. On the other hand, The OARSI score, trabecular spacing, and structural model index of the DM-OA group joint were higher than those of the other two groups.ConclusionsThe glycemic and pancreatic pathological results indicated that the DM-OA model was a simple and reliable model induced by streptozotocin (STZ) and surgery. The results revealed the mechanisms through which diabetes accelerates OA; that is, by damaging and deteriorating the functions of SB, including its microarchitecture, chemical composition, and biomechanical properties.

TLR4 mediates alveolar bone resorption in experimental peri-implantitis through regulation of CD45+ cell infiltration, RANKL/OPG ratio, and inflammatory cytokine production.

The present study was to determine the role of Toll-like receptor 4 (TLR4) signaling in inflammation and alveolar bone resorption using a murine model ofPorphyromonas gingivalis-associated ligature-induced peri-implantitis. Smooth surface titanium implants were placed in the left maxilla alveolar bone 6weeks after extraction of first and second molars in Wild-type (WT) and TLR4-/- (TLR4 KO) mice. Silk ligatures immersed withP. gingivaliswere tied around the implants 4weeks after the implant placement and confirmation of osteointegration. Two weeks after the ligation, bone resorption, osteoclastogenesis, cellular inflammatory responses, and gingival mRNA expression levels of cytokines were assessed by micro-computed tomography, tartrate-resistant acid phosphatase (TRAP) staining, immunobiological examination and Real-time quantitative polymerase chain reaction, respectively. In both WT and TLR4 KO mice, the bone resorption around implants was significantly increased in the P. gingivalis/ligation group compared with control group. In P. gingivalis/ligation group, the levels of bone resorption, TRAP+ cell formation, and gingival CD3+ and CD45+ cell infiltration were significantly decreased in TLR4 KO mice compared with that in WT mice. Receptor activator of nuclear factor-kappa B ligand /osteoprotegerin (RANKL/OPG) ratio was significantly increased after P. gingivalis/ligation treatment in WT mice not in TLR4 KO mice. When comparing the P. gingivalis/ligation group with the respective control group, gingival mRNA expressions of IL-1β, IFN-γ, and 1L-17 were significantly increased in TLR4 KO mice. This study suggests that TLR4 mediates alveolar bone resorption in P. gingivalis associated ligature-induced peri-implantitis through regulation of immune B cell infiltration, RANKL/OPG expression ratio, and differential inflammatory cytokine production.

Compressive force-induced autophagy in periodontal ligament cells downregulates osteoclastogenesis during tooth movement.

Autophagy has recently emerged as a protective mechanism in response to compressive force and an important process in maintenance of bone homeostasis. It appears to be involved in the degradation of osteoclasts, osteoblasts, and osteocytes. The aim of this study was to investigate the role of compressive force-induced autophagy in periodontal ligament (PDL) cells in regulating osteoclastogenesis of orthodontic tooth movement (OTM). An OTM model and compressive force on PDL cells were employed to investigate the expression of autophagy markers in vivo and in vitro, respectively. Autophagosomes and autolysosomes were observed in PDL cells by transmission electron microscope (TEM) and autophagy LC3 double labelling. 3-Methyladenine (3-MA) and rapamycin were respectively used to inhibit and promote autophagy, and the effect of autophagy on osteoclastogenesis was explored via microcomputed tomography, hematoxylin and eosin (H&E) staining, histochemistry of titrate-resistant acid phosphatase, and real-time polymerase chain reaction (RT-PCR) in vivo. Receptor activator of nuclear factor-kappa B ligand/osteoprotegerin (RANKL/OPG) was investigated by RT-PCR and ELISA in vitro. Orthodontic force-induced autophagy was prominent on the pressured side of PDL tissues. Administration of 3-MA downregulated bone density and upregulated osteoclasts, while rapamycin had reverse results in OTM. The autophagy activity increased initially then decreased in PDL cells during compressive force application and responded to light force. In PDL cells, administration of 3-MA upregulated while rapamycin downregulated the RANKL/OPG ratio. Autophagy is activated by compressive force in PDL cells. Besides, it could modulate OTM by negatively regulating osteoclastogenesis and keep bone homeostasis via RANKL/OPG signaling.

Paroxetine and rivastigmine mitigates adjuvant-induced rheumatoid arthritis in rats: Impact on oxidative stress, apoptosis and RANKL/OPG signals

Rheumatoid arthritis (RA) is considered a form of inflammatory autoimmune disease with unknown etiology, but environmental and genetic causes are sharing. T-cells, B-cells, synovial cells, osteoclast, and chondrocytes are the main cell types in RA pathophysiology. The present study aimed to investigate the anti-rheumatic effects of paroxetine, a selective serotonin reuptake inhibitor (SSRI), and rivastigmine, acetyl choline esterase inhibitor (AChEI), in complete Freund's adjuvant (CFA)–induced RA. Adult female rats were categorized into five groups of eight rats each: normal control received vehicles only, RA control received CFA (0.4 ml, s.c) dexamethasone group (1 mg/kg/day, p.o), paroxetine group (10 mg/kg/day, i.p) and rivastigmine group (1 mg/kg/day, i.p). All treatments were administered for 13 consecutive days. Specific rheumatoid marker rheumatoid factor (RF), cartilage oligomeric protein (COMP) and matrix metalloproteinase-3 (MMP-3) were determined. Serum MDA and reduced GSH levels were investigated as oxidative stress biomarkers. IL-6, TNF-α, and monocyte chemotactic protein-1 (MCP-1) were also determined as inflammatory biomarkers. Tissue Receptor activator of nuclear factor kappa-B ligand/osteoprotegerin (RANKL/OPG) expression levels were detected using qRT-PCR. Paroxetine and rivastigmine significantly reduced RF, COMP, MMP-3, IL-6, TNF-α, and MCP-1 serum levels. Tested drugs also significantly reduced serum MDA and increased GSH levels. In addition, paroxetine and rivastigmine attenuated histopathological variations by reducing pannus formation and return synovial fluid near to normal. Administration of paroxetine or rivastigmine normalized caspase-3 and RANKL/OPG in CFA-induced RA.In conclusion, paroxetine and rivastigmine possess antirheumatoid effects in CFA-induced RA which is mediated through antioxidant, anti-inflammatory, antiapoptotic and modulation of RANKL/OPG expression levels.

The synergistic effects of IL-6/IL-17A promote osteogenic differentiation by improving OPG/RANKL ratio and adhesion of MC3T3-E1 cells on hydroxyapatite

ObjectiveIn this study, we evaluated the potential role of IL-6 and/or IL-17A in regulating the OPG/RANKL (osteoprotegerin/receptor activator of nuclear factor kappa b ligand) system of murine osteoblast cell line (MC3T3-E1) cultured on hydroxyapatite (HA). MethodsMC3T3-E1 cells were seeded on HA and treated with recombinant IL-6 or rIL-17A or combination of the two cytokines. Cell proliferation and differentiation activity were measured by MTS and alkaline phosphatase assays respectively. Observation of cell adhesion and proliferation was examined by scanning electron microscopy. Gene and protein expressions were performed on RANKL and OPG using qPCR, Western blot and ELISA. ResultsWe demonstrated that treatment with recombinant IL-17A (rIL-17A) and the combination rIL-6/rIL-17A promoted better adhesion and higher proliferation of cells on HA. Cells treated with rIL-17A and the combination cytokines showed a significant increase in differentiation activity on day 7, 10 and 14 as indicated by ALP activity (p < 0.001). Gene and protein expressions showed significant up-regulation of OPG and ALP (p < 0.001) and down-regulation of RANKL (p < 0.001) expression by all the treated groups. Interestingly, the combination of the two cytokines resulted in a significant increase of OPG/RANKL ratio (p < 0.001). ConclusionThese findings indicated that treatment with the combination of the two cytokines (IL-6/IL-17A) has synergistic effects to promote osteoblastic differentiation but suppress osteoclastogenesis by altering the OPG/RANKL ratio.

Prophylactic supplement with melatonin successfully suppresses the pathogenesis of periodontitis through normalizing RANKL/OPG ratio and depressing the TLR4/MyD88 signaling pathway.

Periodontitis (PD) is an inflammatory disease characterized by gingival inflammation and resorption of alveolar bone. Impaired receptor activator of nuclear factor-kappa B ligand/osteoprotegerin (RANKL/OPG) signaling caused by enhanced production of pro-inflammatory cytokines plays an essential role in the pathogenesis of PD. Considering melatonin possesses significant anti-inflammatory property, this study aimed to determine whether prophylactic treatment with melatonin would effectively normalize RANKL/OPG signaling, depress toll-like receptor 4/myeloid differentiation factor 88 (TLR4/MyD88)-mediated pro-inflammatory cytokine activation, and successfully suppress the pathogenesis of PD. PD was induced in adult rats by placing the ligature at molar subgingival regions. Fourteen days before PD induction, 10, 50, or 100mg/kg of melatonin was intraperitoneally injected for consecutive 28days. Biochemical and enzyme-linked immunosorbent assay were used to detect TLR4/MyD88 activity, RANKL, OPG, interleukin 1β, interleukin 6, and tumor necrosis factor-α levels, respectively. The extent of bone loss, bone mineral intensity, and calcium intensity was further evaluated by scanning electron microscopy, micro-computed tomography, and energy-dispersive X-ray spectroscopy. Results indicated that high RANKL/OPG ratio, TLR4/MyD88 activity, and pro-inflammatory cytokine levels were detected following PD. Impaired biochemical findings paralleled well with severe bone loss and reduced calcium intensity. However, in rats pretreated with melatonin, all above parameters were successfully returned to nearly normal levels with maximal change observed in rats receiving 100mg/kg. As prophylactic treatment with melatonin effectively normalizes RANKL/OPG signaling by depressing TLR4/MyD88-mediated pro-inflammatory cytokine production, dietary supplement with melatonin may serve as an advanced strategy to strengthen oral health to counteract PD-induced destructive damage.

The key role of proinflammatory cytokines, matrix proteins, RANKL/OPG and Wnt/β-catenin in bone healing of hip arthroplasty patients

IntroductionWe still lack understanding of why some implants fail while most remain stable after decades of use. Proinflammatory cytokines, matrix proteins and bone regulating cytokines of the RANKL/OPG (receptor activator of nuclear factor kappa B ligand/osteoprotegerin) and Wnt/β-catenin pathways are mandatory for normal bone repair but their spatial and temporal role in the healing of primary total hip arthroplasties (THA) has not been previously shown. Materials and methodsTwenty-four osteoarthritis patients with one-sided well-fixed primary THA were prospectively monitored during 18years (18Y) with repeated blood samples, clinical variables and radiographs. Eighty-one healthy donors divided in three age- and gender-matched groups and twenty osteoarthritis patients awaiting THA and serving as control of the validity of stored plasma in THA patients, were included. Plasma was analyzed for C-reactive protein (CRP), interleukin (IL)-6, IL-8, IL-1β, tumor necrosis factor (TNF)-α, osteopontin (OPN), secreted protein acidic and rich in cysteine (SPARC/osteonectin), osteocalcin (OC), bone specific alkaline phosphatase (BALP), N-terminal propeptide of collagen type I (P1NP), RANKL, OPG, the Wnt agonistic ligands (Wnt)-1 and Wnt-3a, and the Wnt antagonists sclerostin, Dickkopf (Dkk)-1, Dkk-3, Dkk-4, secreted frizzled related protein (sFRP)-1, sFRP-3 and Wnt inhibitory factor-1 (Wif-1). ResultsInflammatory mediators in arthroplasty patients (CRP, IL-6, OPN) increased significantly on day one after surgery vs preoperative value (PR) and healthy subjects and returned to baseline at 6W. TNF-α did not change relative preoperative level or healthy subjects. SPARC and OC increased in a biphasic fashion with the primary phase beginning shortly after surgery and lasting 3M (SPARC) and 2Y (OC) while the secondary phase peaked at 1Y (SPARC) and 13Y (OC), with both returning to basal level at 15Y. BALP peaked at 3M after surgery with a return to basal level at 2Y followed by a continuous increase from 5Y until 18Y. P1NP increased immediately after surgery and returned to basal level at 6W followed by a new peak at 10Y returning to basal at 13Y. IL-8 and IL-1β peaked at 5Y post-THA and returned to basal level at 10Y. RANKL/OPG and Wnt/β-catenin remained at preoperative levels until 5Y post-THA when a sustained increase in OPG level, paralleled by a sustained decrease in sclerostin, started and lasted until 18Y. Despite a strong increase by RANKL at 13Y, the OPG/RANKL-ratio remained high between 5Y and 18Y. Dkk-1 and sFRP-1 remained at basal level until 5Y followed by a peak at 7Y and a return to basal level at 15Y. Similarly, RANKL increased after 5Y, peaked at 13Y and returned to basal levels at 18Y, thus coinciding with Wnt-1. In contrast, Wnt3a, Dkk-3, Dkk-4, sFRP-3 and Wif-1 did not differ from preoperative levels or healthy subjects during the course of the follow-up. ConclusionThe primary peak of proinflammatory cytokines involved in the initiation of bone healing after trauma is in line with previous results. The primary phase of increased matrix proteins, P1NP and BALP paralleled by RANKL, OPG and Wnt/β-catenin remaining at preoperative level until 5Y, support a strong formation of mineralized matrix and to a lesser degree bone during this phase. The secondary proinflammatory peak at 5Y is likely a trigger of coupled bone remodeling and neosynthesis as it is followed by increased levels of the bone anabolic turnover marker, BALP, and mediators of the RANKL/OPG and Wnt/β-catenin pathways. A continuous increase by OPG level and the bone turnover marker, BALP, lasting from 5Y until 18Y and paralleled by a similar decrease in sclerostin level support their being key regulators of bone anabolism, whereas the transient and opposed activities of RANKL, Wnt-1, Dkk-1 and sFRP-1 serve as fine tuning tools during the coupled remodeling phase.

Osteoclast-activating system in cosmonauts after long-term international space station missions

The results of studying the system of the osteoprotegerin/receptor activator of nuclear factor kappa-B ligand (OPG/RANKL) in 22 Russian cosmonauts after long-term (124–199 days) International Space Station missions are presented. It was found that early readaptation period is associated with changes in the serum concentrations of OPG and RANKL and the production rate of OPG and RANKL in phytohemagglutinin (PHA)-stimulated and non-stimulated mononuclear cells of peripheral blood in vitro. The extent of these changes varied in different subjects. Our data suggest that the OPG/RANKL cytokine system takes part in the processes of bone remodeling in cosmonauts after long-term missions.

GPR40 mediates potential positive effects of a saturated fatty acid enriched diet on bone.

The stimulation of the free fatty acid receptor G-protein coupled receptor (GPR) 40 by GW9508 prevents bone loss by inhibiting osteoclast activity, both in vitro and in vivo. Here, we questioned whether the stimulation of the GPR40 receptor by dietary fatty acids may lead to the same beneficial effect on bone. We investigated (i) the impact of a fatty acid enriched diet (high-fat diet [HFD]) on bone health in C57/BL6 female mice depending on (ii) the estrogen status (ovariectomy) and (iii) the genotype (GPR40+/+ or GPR40-/- ). Bone mineral density (BMD), body composition, weight, inflammation and bone remodeling parameters were monitored. HFD decreased BMD in HFD-SH-GPR40+/+ mice but OVX failed to further impact BMD in HFD-OVX-GPR40+/+ mice, while additional bone loss was observed in HFD-OVX-GPR40-/- animals. These data suggest that when stimulated by fatty acid enriched diets GPR40 contributes to counteract ovariectomy-induced bone alteration. The sparing effect is supported by the modulation of both the osteoprotegerin/receptor activator of nuclear factor kappa-B ligand (OPG/RANKL) ratio in blood stream and the expression level of inflammatory markers in adipose tissues. Bone preservation by GPR40 stimulation is dependent on the presence of long-chain saturated fatty acids. GPR40 contributes to counter ovariectomy-induced bone loss in a context of saturated fatty acid enrichment.

Bone Regeneration Is Promoted by Orally Administered Bovine Lactoferrin in a Rabbit Tibial Distraction Osteogenesis Model.

Lactoferrin, an iron-binding glycoprotein which belongs to the transferrin family, has been shown to promote bone growth. However, reports regarding effects of lactoferrin on bone regeneration during distraction osteogenesis are limited. Our study was designed to investigate the effect of bovine lactoferrin treatment on bone formation of the distracted callus. We asked whether bovine lactoferrin enhances bone formation of the distraction callus as determined by (1) radiographic and histologic appearances; (2) dual-energy x-ray absorptiometry (DXA) analysis of bone mineral composition and bone mineral density; (3) micro-CT measures of trabecular architecture; and (4) biomechanical strength of the healing bone. Additionally, serology, reverse transcription (RT)-PCR, and immunohistochemistry were used to explore the possible mechanisms of bovine lactoferrin use on bone formation during distraction osteogenesis. Unilateral tibial osteodistraction was performed on 80 New Zealand White rabbits with a distraction rate of 1 mm per day for 10 days. Animals then were divided randomly into two groups: (1) vehicle and (2) bovine lactoferrin. At 4 and 8 weeks after completion of distraction, the animals were sacrificed. Lengthened tibias and serum samples were obtained and subjected to radiologic, DXA, micro-CT, histologic, and biomechanical examinations, and serum, RT-PCR and immunohistochemical analyses. Radiologic, DXA, micro-CT, histologic, and biomechanical examinations indicated that bovine lactoferrin treatment not only accelerated bone formation at early stages of distraction osteogenesis but also promoted bone consolidation at late stages. The ultimate force of the distracted calluses was increased by 37% (118.8 ± 6.65 N in the lactoferrin group and 86.5 ± 5.47 N in the vehicle group; p < 0.001) and 84% (384.8 ± 18.4 N in the lactoferrin group and 209.0 ± 15.2 N in the vehicle group; p < 0.001) at 4 and 8 weeks, respectively. Moreover, serum analysis showed that bovine lactoferrin treatment significantly increased serum levels of bone alkaline phosphatase and decreased serum levels of tartrate resistant acid phosphatase 5b. In addition, RT-PCR and immunohistochemical analyses suggested that bovine lactoferrin treatment induced a lower receptor activator of nuclear factor-kappaB (RANK) ligand/osteoprotegerin (RANKL/OPG) ratio in the distracted callus. The results of our study suggest that bovine lactoferrin treatment could promote bone regeneration during distraction osteogenesis in the rabbit. The results indicate that the OPG/RANKL/RANK system might be a major mechanism for increased bone formation and decreased bone resorption in distraction osteogenesis with bovine lactoferrin treatment. Oral administration of bovine lactoferrin may provide a feasible approach for promoting osteogenesis during distraction osteogenesis.

Kirenol stimulates osteoblast differentiation through activation of the BMP and Wnt/β-catenin signaling pathways in MC3T3-E1 cells

Kirenol has been reported to possess anti-oxidant, anti-inflammatory, anti-allergic, anti-adipogenic, and anti-arthritic activities; however, its effect on osteoblast differentiation has not yet been reported. The aim of the present study was to evaluate the effect of kirenol on osteoblast differentiation through activation of the bone morphogenetic protein (BMP) and Wnt/β-catenin signaling pathways in MC3T3-E1 cells. Kirenol markedly promoted alkaline phosphatase (ALP) activity and mineralization. Kirenol not only increased the expression of osteoblast differentiation markers, such as ALP, type I collagen (ColA1), and osteopontin (OPN), but also increased the expression of osteoprotegerin/receptor activator of nuclear factor kappa B ligand (OPG/RANKL) ratio. The effects of kirenol on osteoblast differentiation were accompanied by stimulating the expression of the BMP and Wnt/β-catenin signaling pathways, including BMP2, runt-related transcription factor 2 (Runx2), osterix (Osx), low density lipoprotein receptor related protein 5 (LRP5), disheveled 2 (DVL2), β-catenin, cyclin D1 (CCND1), and phosphorylated glycogen synthase kinase 3β (GSK3β). In addition, kirenol up-regulated the expression of β-catenin, CCND1, ALP, and ColA1 which were down-regulated by siRNA knockdown of β-catenin. Overall, these results demonstrate that kirenol is capable of promoting osteoblast differentiation in MC3T3-E1 cells through activation of the BMP and Wnt/β-catenin signaling pathways, suggesting that it is a potential candidate target for treating or preventing osteoporosis.

Effects of Huogu I Formula (活骨I方) on correlated factors of bone regeneration in chickens with steroid-induced necrosis of femoral head

To study the mechanism of Huogu I formula (I) in treating osteonecrosis of femoral head. Forty-eight healthy female Leghorn chickens were randomly divided into control group, model group and Huogu I group, and each group consisted of 16 chickens. At the meantime of model establishment, chickens of the Huogu I group were administrated with decoction, while the model and control group with distilled water by gavage. At the 8th and 16th week after medication, blood samples were obtained for blood lipid detection while both sides of femoral head were harvested for the rest of examinations. Specifically, expressions of bone morphogenetic protein-2 (BMP2), transforming growth factor beta1 (TGFβ(1)), Smad4 and Smad7 were evaluated by immunohistochemistry, while expression of osteoprotegerin/receptor activator of nuclear factor kappaB ligand (OPG/RANKL) mRNA was detected by in situ hybridization. Compared with the control group, serum levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in the model group rose significantly. Positive cell counting of BMP2, TGFβ(1), Smad4 and OPG in femoral head of the model group dropped prominently. Positive cell counting of Smad7 and RANKL increased dramatically. In contrast with the model group, levels of TC, TG and LDL-C in Huogu I group reduced significantly. Positive cell counting of BMP2, TGFβ(1), Smad4 and OPG in femoral head of the Huogu I group increased prominently. Indices of Smad7 and RANKL both decreased significantly. Especially at the 8th week, these variations were more significant. Huogu I formula is effective in promoting repair of necrotic femoral head by regulating the expressions of BMP2, TGFβ(1), Smads and OPG/RANKL of osteoclast in femoral head.

Protective Effect of Metformin on Periapical Lesions in Rats by Decreasing the Ratio of Receptor Activator of Nuclear Factor Kappa B Ligand/Osteoprotegerin

IntroductionMetformin, one of the antihyperglycemic agents commonly used for the treatment of type 2 diabetes, was shown to inhibit osteoclast formation. The current study aimed to investigate the effects of systemically administered metformin on alveolar bone resorption and on the ratio of receptor activator of nuclear factor kappa B ligand/osteoprotegerin (RANKL/OPG) in rats subjected to experimental periapical lesions. MethodsForty adult male Wistar rats were divided equally into control and experimental groups, and the pulp chambers of their mandibular first molars were exposed to the oral environment to induce periapical lesions. The experimental group received daily intramuscular injections of metformin at 40 mg/kg doses, whereas the control group received only the saline vehicle. The injections were initiated 1 day before the periapical lesion induction and then were continued daily throughout the entire experimental period. Two or 4 weeks after pulp exposure, the rats were killed, and the mandibles were prepared for histologic analysis, enzyme histochemistry, immunohistochemistry, and immunofluorescence. ResultsThe number of RANKL-positive and tartrate-resistant acid phosphatase (TRAP)–positive cells in the metformin-treated groups decreased on day 14, whereas the number of OPG-positive cells increased on day 28. The periapical bone loss area in the metformin-treated group significantly decreased on day 28 compared with the control group. ConclusionsMetformin inhibits the periapical lesions possibly by lowering the RANKL/OPG ratio, subsequently reducing the number of osteoclasts and bone resorption areas.

Charcot osteoarthropathy: one disease, two presentations

Charcot osteoarthropathy or Charcot foot is a disabling complication of diabetes and is associated with poor prognosis and high mortality. Its pathogenesis is not fully understood and its treatment is at best symptomatic. Furthermore, it is not known whether there is a specific type of neuropathy which affects osteoclastic activity, and thereby leads to reduction of bone mineral density and the development of Charcot osteoarthropathy. Recently it has been proposed that there is a difference in the presentation of Charcot osteoarthropathy between type 1 and type 2 diabetes. This article reviews the link between underlying osteopenia, abnormal biomechanical forces and type of neuropathy, and their varying interaction in the pathogenesis of Charcot osteoarthropathy in type 1 and type 2 diabetes. Further attention is drawn to the newly discovered osteoprotegerin/receptor activator of nuclear factor kappaB ligand (OPG/RANKL) cytokine system, which controls bone resorption. Increased osteoclastic activity in the acute Charcot foot may be associated with altered expression of OPG/RANKL signaling pathway and modulation of the OPG/RANKL equilibrium in Charcot osteoarthropathy may provide additional therapeutical option to manage this difficult condition. Biomedical Reviews 2005; 16: 43-48.