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 / 中国骨伤

OBJECTIVE@#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).@*METHODS@#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.@*RESULTS@#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).@*CONCLUSION@#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.

Beneficial effects of Cuscuta chinensis extract on glucocorticoid-induced osteoporosis through modulation of RANKL/OPG signals

Cuscuta chinensis Lam. (Convolvulaceae) is an important herbal medicine widely used to improve sexual function, treat osteoporosis, and prevent aging, and has been reported to exhibit anti-osteoporotic effects in vitro. However, the activity of Cuscuta chinensis Lam. on glucocorticoid-induced osteoporosis still remains unclear. The present study aimed to assess the protective effect and the underlying mechanism of action of Cuscuta chinensis extract (CCE) against glucocorticoid-induced osteoporosis in vivo. Sprague-Dawley rats were randomly divided into four groups as follows: control group, osteoporosis group, and 2 CCE-treated osteoporosis groups (100 mg·kg-1·day-1). Blood samples and femur bones were collected for immunohistochemistry, biochemical, mRNA expression, and western blot analysis. HPLC analysis revealed that chlorogenic acid, quercetin, and hyperin were the major constituents of CCE. The results indicated that CCE increased bone length, bone weight, and bone mineral density and suppressed dexamethasone (DEX)-induced reduction in body weight. In addition, TRAP staining indicated that CCE reduced osteoclasts in DEX-induced osteoporosis rats. Mechanistically, CCE treatment alleviated the increase of bone resorption markers and the decline of osteogenic markers, which might be partially mediated by regulation of RANKL/OPG and RunX2 pathways. These results suggest that CCE showed promising effects in the protection against glucocorticoid-induced osteoporosis through protecting osteoblasts and suppressing osteoclastogenesis.

Chronic Periodontitis and RANKL/OPG Ratio in Peri-Implant Mucosae Inflammation

Abstract tHistory of chronic periodontitis (CP) is a risk factor for oseointegration failure. The osteoclastogenesis system (RANK, RANKL and OPG) is critical for bone homeostatic control. We investigated the levels of OPG and RANKL in peri-implant tissues from volunteers with and without a history of CP and their association with mucosae inflammation. This is a single-blind case-contro study. Diagnosis of a history of CP and peri-implant examination was performed on 46 volunteers, divided into control (without history of CP, n=26) and CP group (with history of CP, n=20). Gingival biopsies were harvested during implant exposure. Quantitative PCR evaluated OPG/RANKL mRNA expressions. OPG and RANKL proteins were analyzed by western blot and immunohistochemistry assay. The chi-square test analyzed the significance of nominal variables between groups while continuous variables were analyzed by T-test or Mann-Whitney test, after Shapiro-Wilk test evaluation. The 2-ΔΔCT Livak method calculation evaluated the gene expression. Values of p<0.05 were considered statistically significant. Volunteers with CP history had 23 times higher chance of developing mucosae inflammation. High mucosae levels of RANKL (p=0.04) and RANKL/OPG (p=0.001) mRNA expressions were observed in CP group. CP volunteers showed increased RANKL protein levels in opposition to decreased OPG expression. Even without active periodontitis, volunteers with a history of CP had elevated gingival levels of RANKL/OPG and higher correlation with peri-implant mucosae inflammation and implant loss.

Resumo A história de periodontite crônica (CP) é um fator de risco para falhas na osseointegração. O sistema de osteoclastogênese (RANK, RANKL e OPG) é crucial para o controle da homeostase óssea. O objetivo deste estudo foi investigar os níveis de OPG e RANKL no tecido peri-implantar de voluntários com e sem histórico de CP e sua associação com inflamação da mucosa. Este é um estudo tipo caso-controle. O exame para diagnóstico de CP e na região peri-implantar foi realizado em 46 voluntários, divididos em controle (sem história CP, n=26) e grupo CP (com histórico de CP, n=20). Descartes gengivais foram obtidos durante a exposição do implante. PCR quantitativo avaliou a expressão do RNAm de OPG/RANKL. As proteínas OPG e RANKL foram analisadas por western blot e imunohistoquímica. O teste do qui-quadrado analisou a significância entre as variáveis nominais enquanto as variáveis contínuas foram analisadas pelo teste-t e Mann-Whitney, após o teste de Shapiro-wilk. O método do Livak 2--ΔΔCT avaliou a expressão gênica. Os voluntários com CP apresentaram 23 vezes mais chances de desenvolver inflamação da mucosa. Expressão elevada no RNAm de RANKL (p=0.04) e RANKL/OPG (p=0.001) foram observadas no grupo CP. Voluntários com CP mostraram aumento dos níveis da proteína RANKL em contraste com diminuída expressão de OPG. Mesmo sem periodontite ativa, voluntários com histórico de CP apresentaram elevado nível gengival de RANKL/OPG e alta correlação com inflamação peri-implantar e perda do implante.

Evolution of Periodontal Disease: Immune Response and RANK/RANKL/OPG System

Abstract The aim of this study was to evaluate markers of bone loss and immune response present in evolution of periodontal disease. One hundred and two Wistar rats were divided into three animals groups: PD0, without ligation and PD15 days and PD60 days, submitted to ligation placement with a sterile 3-0 silk cord in the cervical region of the upper first molar on both sides. Samples were obtained from the gingival tissue for histomorphometric analysis, immunohistochemical analysis of RANK, RANKL, OPG, characterization of the inflammatory infiltrate, quantification of nitric oxide, MCP-1, RANTES, IP10 chemokines, and expression of the TGF-b1, VEG, and bFGF. The number of inflammatory cells in gingival tissue was higher in PD60 samples. The collagen content and the area occupied by birefringent collagen fibers were lower for PD60. Differential leukocyte counting showed that there was a significantly higher polymorphonuclear influx in group PD15, while PD60 showed a greater number of lymphocytes. PD60 showed higher RANTES, IP-10, MCP-1 gene transcripts, as well as a higher nitric oxide concentration. Clinical evaluation revealed that the PD60 group presented an increase in furcal area. In conclusion, in this animal model the increase of RANK/RANKL and HGF markers is related to a specific immune response, and probably contributed to the evolution of periodontal disease. Investigating the effect of these biomarkers can help in targeted therapy for bone resorption, since blocking these can inhibit bone loss.

Resumo Este estudo avaliou marcadores de perda óssea e da resposta imune presentes na evolução da doença periodontal. Cento e dois ratos Wistar foram divididos em três grupos de animais: PD0, sem ligadura e PD15 dias e PD60 dias, submetidos a colocação de ligadura com um fio de seda estéril 3-0 na região cervical do primeiro molar superior em ambos os lados. Foram obtidas amostras de tecido gengival para análise histomorfométrica, análises imunohistoquímicas de RANK, RANKL, OPG, caracterização do infiltrado inflamatório, quantificação de óxido nítrico, expressão de quimiocinas MCP-1, RANTES, IP10 e do TGF-b1, VEGF e bFGF . O número de células inflamatórias no tecido gengival foi maior nas amostras PD60. O teor de colágeno na área ocupada pelas fibras de colágeno birrefringentes foram menores para PD60. A contagem diferencial de leucócitos mostrou que houve um influxo polimorfonuclear significativamente maior no grupo PD15, enquanto que PD60 mostrou número maior de linfócitos. PD60 apresentou transcritos de genes RANTES, IP-10, MCP-1 mais elevados, bem como uma maior concentração de óxido nítrico. A avaliação clínica revelou que o grupo PD60 apresentou aumento da área óssea exposta na região da furca. Em conclusão, neste modelo animal o aumento dos marcadores RANK/RANKL e HGF está relacionado a uma resposta imunológica específica e provavelmente contribuiu para a evolução da doença periodontal. Investigar o efeito destes biomarcadores pode ajudar na terapia dirigida para a reabsorção óssea, uma vez que bloquear estes pode inibir a perda óssea.

The effects of chitosan oligosaccharides on OPG and RANKL expression in a rat osteoarthritis model

Abstract Purpose: To investigate the effect of chitosan oligosaccharides (COS) against osteoarthritis (OA) and preliminarily discuss the osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL) and RANK expression in a rat OA model. Methods: Thirty-six 6-week-old Male SD rats were randomly divided into three groups: sham-operated group(CON), OA-induction group(OA), COS intervention group(n=12/group). At 4 weeks after the operation, COS (50 ul) intervention weekily for consecutive 5 weeks. The OA and CON groups received an injection of 50 ul physiological saline. At death, 11 weeks following surgery, cartilage was harvested and total RNA and protein were extracted. Both the morphological changes of the cartilage were observed and harvested the total RNA and protein. Meanwhile, the expression of OPG, RANKL and RANK in cartilage were determined. Results: The expression of OPG and RANKL were both enhanced in the cartilage of the OA model. Compared with the OA group, COS treatment improved the cartilage damage (both extent and grade). Furthermore, the COS group showed highly OPG and lower RANKL. Simultaneously, COS treatment upregulated the ratio of OPG/RANKL and downregulated the RANKL/RANK. Conclusion: Chitosan oligosaccharides may be used as a unique biological agent to prevent and treat osteoarthritis, and this effect is associated with modulation of the expression of osteoprotegerin and receptor activator of NF-κB ligand.

TLR2 Signaling Pathway in Alveolar Bone Osteogenesis Induced by Aloe vera and Xenograft (XCB)

Abstract The aim of this study was to find the role of TLR2 signaling pathway in reducing osteoclast activity and promoting osteoblast growth by inducing a combination of Aloe vera and cancellous bovine xenograft (XCB) into dental extraction socket. Forty-eight Cavia cobayas were used. They were divided into eight groups (n=6). For control group, their mandibular incisors were extracted and filled with PEG. For treatment groups, they were extracted and filled with XCB, Aloe vera and the combination of Aloe vera and XCB. The first four groups were sacrificed after 7 days and the other groups after 30 days. Immunohistochemistry and histopathology examination were conducted to examine TLR2, TNFa, OPG, collagen-1, and the osteoblast and osteoclast expressions. The expressions of TLR2, OPG and Collagen-1, as well as the number of osteoblast were increased. Meanwhile, the expressions of TNFa and osteoclast were decreased. The study finding was that TLR2 signaling pathway influenced alveolar bone osteogenesis process by reducing osteoclast activity and stimulating osteoblast growth induced by the combination of Aloe vera and XCB.

Resumo O objetivo deste estudo foi investigar o papel da via de sinalização de TLR2 na redução da atividade osteoclástica e na promoção do crescimento de osteoblastos, induzindo uma combinação de Aloe vera e enxerto de osso esponjoso bovino (EOEB) em alvéolo de extração dentária. Quarenta e oito Cavia cobayas foram utilizados e divididos em 8 grupos (n = 6). Para o grupo de controle, seus incisivos mandibulares foram extraídos e preenchidos com polietilenoglicol (PEG). Para grupos de tratamento, os dentes foram extraídos e preenchidos com EOEB, Aloe vera e a combinação de Aloe vera e EOEB. Os primeiros quatro grupos foram sacrificados após 7 dias e os outros grupos após 30 dias. As análises de imunohistoquímica e histopatologia foram realizada para examinar TLR2, TNFa OPG, colágeno-1 e as expressões de osteoblastos e osteoclastos. Houve maior expressão de TLR2, FGF2, OPG e colágeno-1, bem como maior número de osteoblastos. Enquanto isso, a expressão de TNFa e osteoclastos estava diminuída. O principal achado do estudo foi que a via de sinalização de TLR2 influenciou o processo de osteogênese do osso alveolar, reduzindo a atividade dos osteoclastos e estimulando o crescimento de osteoblastos induzido pela combinação de Aloe vera e EOEB.

Effects of IL-10 and glucose on expression of OPG and RANKL in human periodontal ligament fibroblasts

The effects of interleukin-10 (IL-10) and glucose on mRNA and protein expression of osteoprotegerin (OPG), and its ligand, receptor activator of nuclear factor-κB ligand (RANKL), were investigated in human periodontal ligament fibroblasts (HPDLFs). Primary HPDLFs were treated with different concentrations of IL-10 (0, 1, 10, 25, 50, and 100 ng/mL) or glucose (0, 5.5, 10, 20, 30, and 40 mmol/L). Changes in mRNA and protein expression were examined using the reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. After IL-10 treatment, mRNA and protein levels of OPG were increased, while mRNA and protein levels of RANKL were decreased (P<0.05), both in a concentration-dependent manner. Glucose stimulation had the opposite concentration-dependent effect to that of IL-10 on OPG and RANKL expression. IL-10 upregulated OPG expression and downregulated RANKL expression, whereas high glucose upregulated RANKL and downregulated OPG in HDPLFs. Abnormal levels of IL-10 and glucose may contribute to the pathogenesis of periodontal disease.

Elevated serum osteoprotegerin levels in women: friend or foe? / Níveis séricos elevados de osteoprotegerina na mulher: aliado ou inimigo?

SUMMARY Introduction: osteoprotegerin has emerged as a new candidate for the treatment of osteoporosis. However, high levels of osteoprotegerin have been linked to vascular calcification, an independent and well-defined risk factor for cardiovascular disease (CVD) and mortality. Thus, the action of osteoprotegerin in these situations has been questioned. Objective: to evaluate the effect of osteoprotegerin (OPG) on the human body, especially in bone tissue and in vascular diseases. Methods: the scientific databases consulted were PubMed-Medline and Cochrane, using keywords (MeSH terms) grouped into the following syntaxes: (Osteoprotegerin OR Osteoclastogenesis Inhibitory Factor OR Receptors, Tumor Necrosis Factor, Member 11b OR Tumor Necrosis Factor Receptor Superfamily, Member 11b OR FDCR-1 Protein OR FDCR 1 Protein OR OCIF Protein OR Follicular Dendritic Cell-Derived Receptor-1) AND (Bones AND Bone OR Bones AND Bone Tissue OR Bones OR Bone Tissue OR Cardiovascular Diseases). Results: Osteoprotegerin is present in various organs and binds to two ligands: nuclear factor kB (RANKL) related to the differentiation of osteoclasts, and tumor necrosis factor related to the apoptosis-inducing ligand (TRAIL). OPG inhibits the regulation effects of nuclear factor kB on inflammation and on the skeletal and vascular systems, preventing the apoptosis induced by TRAIL, being related to the preservation of bone tissue. Conclusion: a deeper knowledge of the mechanisms involved in the association between OPG serum levels, bone integrity and cardiovascular disease can provide important data for future therapeutic interventions.

RESUMO Introdução: a osteoprotegerina (OPG) tem surgido como uma nova candidata para o tratamento da osteoporose; no entanto, níveis elevados de OPG têm sido relacionados à calcificação vascular, um fator de risco independente e bem definido para doença cardiovascular (DCV) e mortalidade. Assim, a ação da OPG nessas situações tem sido questionada. Objetivo: avaliar a ação da OPG no corpo humano, em especial no tecido ósseo e nas doenças vasculares. Métodos: as bases de informação científica consultadas foram PubMed-Medline e Cochrane, utilizando-se palavras-chave (MeSH terms) agrupadas nas seguintes sintaxes: (Osteoprotegerin OR Osteoclastogenesis Inhibitory Factor OR Receptors, Tumor Necrosis Factor, Member 11b OR Tumor Necrosis Factor Receptor Superfamily, Member 11b OR FDCR-1 Protein OR FDCR 1 Protein OR OCIF Protein OR Follicular Dendritic Cell-Derived Receptor-1) AND (Bones AND Bone OR Bones AND Bone Tissue OR Bones OR Bone Tissue OR Cardiovascular Diseases). Resultados: a OPG está presente em vários órgãos e une-se a dois ligantes: o fator nuclear kB (RANKL), relacionado com a diferenciação dos osteoclastos, e o fator de necrose tumoral, relacionado ao ligante indutor de apoptose (TRAIL). Assim, a OPG inibe os efeitos da regulação do fator nuclear kB na inflamação e nos sistemas esquelético e vascular, prevenindo a apoptose induzida pelo TRAIL, estando relacionada com a preservação do tecido ósseo. Conclusão: um conhecimento mais aprofundado sobre os mecanismos envolvidos na associação entre os níveis séricos da OPG, integridade óssea e doenças cardiovasculares podem proporcionar dados importantes para futuras intervenções terapêuticas.

Static compression regulates OPG expression in periodontal ligament cells via the CAMK II pathway

ABSTRACT Objective This study aimed to investigate the potential role of CAMK II pathway in the compression-regulated OPG expression in periodontal ligament cells (PDLCs). Material and Methods The PDL tissue model was developed by 3-D culturing human PDLCs in a thin sheet of poly lactic-co-glycolic acid (PLGA) scaffolds, which was subjected to static compression of 25 g/cm2 for 3, 6 and 12 h, with or without treatment of KN-93. After that, the expression of OPG, RANKL and NFATC2 was investigated through real-time PCR and western blot analysis. Results After static compression, the NFATC2 and RANKL expression was significantly up-regulated, while partially suppressed by KN-93 for 6 and 12 h respectively. The OPG expression was significantly down-regulated by compression in 3 h, started to elevate in 6 h, and significantly up-regulated in 12 h. The up-regulation after 12 h was significantly suppressed by KN-93. Conclusions Long-term static compression increases OPG expression in PDLCs, at least partially, via the CAMK II pathway.

Localización de OPG en la pulpa dental del Ratón Albino Suizo / OPG Location in dental pulp of Albino Swiss Mice

En condiciones fisiológicas, el órgano dentinopulpar no se encuentra asociado a procesos de remodelación y resorción. La Osteoprotegerina (OPG) es una molécula del Factor de Necrosis Tumoral (TNF) que participa en la diferenciación y activación de osteoclastos, por lo que su papel en el control del proceso de reabsorción radicular interna debe ser determinado. Se tomaron secciones histológicas de 5 µm tomadas de primeros molares de 8 hemimandíbulas de ratón y fueron sometidas a proceso de inmunohistoquímica con la técnica de complejo avidina-biotina peroxidasa para la detección de OPG. Se identificó la presencia de OPG en las regiones central y periférica de la pulpa de ratones de 4 y 12 semanas. La pulpa de ratones albino suizo mostró presencia diferencial de OPG en las zonas central y periférica

Under physiological conditions, the dentinopulpar complex is not associated with remodeling and resorption. Osteoprotegerin (OPG) is a TNF molecule associated with osteoclast activation and differentiation. Therefore, OPG role in internal root resorption must be determined. We obtained histological sections of 5 µm in first molars from 8 hemimandibles of mice, which were subjected to the avidin-biotin peroxidase complex immunohistochemical procedure for detection of OPG. Differential OPG presence was identified in central and peripheral pulp in dental pulp of 4 and 12 week-old mice

Effect of triiodothyronine on the maxilla and masseter muscles of the rat stomatognathic system

The maxilla and masseter muscles are components of the stomatognathic system involved in chewing, which is frequently affected by physical forces such as gravity, and by dental, orthodontic and orthopedic procedures. Thyroid hormones (TH) are known to regulate the expression of genes that control bone mass and the oxidative properties of muscles; however, little is known about the effects of TH on the stomatognathic system. This study investigated this issue by evaluating: i) osteoprotegerin (OPG) and osteopontine (OPN) mRNA expression in the maxilla and ii) myoglobin (Mb) mRNA and protein expression, as well as fiber composition of the masseter. Male Wistar rats (~250 g) were divided into thyroidectomized (Tx) and sham-operated (SO) groups (N = 24/group) treated with T3 or saline (0.9 percent) for 15 days. Thyroidectomy increased OPG (~40 percent) and OPN (~75 percent) mRNA expression, while T3 treatment reduced OPG (~40 percent) and OPN (~75 percent) in Tx, and both (~50 percent) in SO rats. Masseter Mb mRNA expression and fiber type composition remained unchanged, despite the induction of hypo- and hyperthyroidism. However, Mb content was decreased in Tx rats even after T3 treatment. Since OPG and OPN are key proteins involved in the osteoclastogenesis inhibition and bone mineralization, respectively, and that Mb functions as a muscle store of O2 allowing muscles to be more resistant to fatigue, the present data indicate that TH also interfere with maxilla remodeling and the oxidative properties of the masseter, influencing the function of the stomatognathic system, which may require attention during dental, orthodontic and orthopedic procedures in patients with thyroid diseases.