RANK, RANKL and OPG expressions in a permanent molar with a replacement resorption

The aim of this study was to explore the expression of RANK, RANKL and OPG during ankylosis. Structural details and immunohistochemical investigations of the expression of RANK, RANKL and OPG in an extracted secondary retained permanent molar of a 12-year-old girl are reported. Woven and lamellar bones were observed in the thickness of the remodeled dental wall and a tertiary dentin was noticed around the pulp cavity. The resorbing multinucleated cells expressed TRAP and RANK but few of them also expressed RANKL. Both odontoblasts and osteoblasts expressed TRAP and RANK, but the expression of RANKL was limited to osteoblasts. OPG remained undetected. The present case reveals unusual expression of RANKL in the resorbing cells, TRAP and RANK in both osteoblasts and odontoblasts, and a failure of detection of OPG. These proteins could be involved in the pathogenesis of tooth ankylosis.     

Periapical tissue reactions to calcium hydroxide and MTA after external root resorption as a sequela of delayed tooth replantation

Clinical experience has shown that most avulsed teeth are replanted after a long extra-alveolar time and dry or inadequate wet storage, causing necrosis of periodontal ligament cells. This condition invariably leads to development of external root resorption, leaving the filling material in contact with the periapical connective tissues. In this study, the periapical tissue reactions to calcium hydroxide (CH) and mineral trioxide aggregate (MTA) were evaluated after occurrence of external root resorption as an expected sequela of delayed tooth replantation. Twenty male Wistar rats (Rattus norvegicus, albinus) had their right upper incisor extracted and maintained in dry storage for 60 min. Then, the dental papilla, enamel organ, pulp tissue, and periodontal ligament were removed, and the teeth were immersed in a 2% acidulated phosphate sodium fluoride solution, pH 5.5, for 10 min. The teeth were randomly assigned into two groups (n = 10), in which the canals were filled with either a CH and saline paste (CH group) or MTA (MTA group). The sockets were irrigated with saline, and the teeth were replanted. After 80 days, it was possible to observe large areas of replacement root resorption and some areas of inflammatory root resorption in both groups. More severe inflammatory tissue reaction was observed in contact with calcium hydroxide compared with the mineral trioxide aggregate. New bone formation was more intense at the bottom of the socket in the MTA group. In conclusion, as far as periapical tissue compatibility is concerned, intracanal MTA can be considered as a viable option for root canal filling in delayed tooth replantation, in which external root resorption is an expected sequela.     

三氧化矿物凝聚体对乳、恒牙牙髓细胞增殖和分化影响的比较

目的 对比三氧化矿物凝聚体（MTA）和氢氧化钙对人乳、恒牙牙髓细胞增殖和分化的影响。方法 采用甲基噻唑基四唑（MTT）法检测牙髓细胞生长增殖变化;实时荧光定量聚合酶链反应（PCR）检测碱性磷酸酶（ALP）、牙本质涎磷蛋白（DSPP）、骨保护因子（OPG）、核因子κB受体活化因子配体（RANKL）基因的表达。结果 氢氧化钙组乳、恒牙牙髓细胞增殖均显著低于对照组（P<0.01）,MTA组乳、恒牙牙髓细胞增殖均高于对照组（P<0.01）。实时荧光定量PCR检测结果显示,乳牙氢氧化钙组ALP、DSPP和OPG的表达显著低于对照组（P<0.01）,MTA组上述因子的表达显著高于对照组（P<0.01）;氢氧化钙组RANKL的表达显著高于对照组（P< 0.01）,MTA组RANKL的表达与对照组差异无统计学意义（P>0.05）。恒牙牙髓细胞氢氧化钙组ALP和DSPP的表达与对照组相比无显著差异（P>0.05）,MTA组ALP和DSPP的表达显著增加（P<0.01）;氢氧化钙组和MTA组OPG、RANKL的表达与对照组无显著差异（P>0.05）。结论 MTA比氢氧化钙更适合做乳牙和恒牙的盖髓剂,其优势在乳牙可能更为明显。     

Effect of delayed calcium hydroxide treatment on periodontal healing in contaminated replanted teeth

Abstract – The effect of delayed intracanal calcium hydroxide treatment on experimentally induced extensive inflammatory root resorption in monkeys was studied. A significant shift from inflammatory resorption to ankylosis was noted following calcium hydroxide treatment. Furthermore, calcium hydroxide treatment appeared to change the pattern of ankylosis over time, although the total ankylotic area remained the same. Ankylosis preceded by root resorption (replacement resorption) increased, while ankylosis not associated with root resorption decreased. It was concluded that intracanal calcium hydroxide treatment of teeth with compromised PDM may cause unnecessary replacement resorption if left in the root canal for a long time or changed repeatedly.     

抑制核因子-κB受体活化因子及其配体通路治疗牙周炎的研究进展

核因子-κB受体活化因子(RANK)及其配体(RANKL)在牙周炎患者的牙槽骨吸收中具有重要的作用,抑制RANKL/RANK通路,可有效抑制破骨细胞的分化和激活,从而抑制牙槽骨的吸收。骨保护蛋白(OPG)可和RANKL结合,干扰RANKL和RANK的结合,从而防止骨组织的过度破坏。本文就RANKL/RANK/OPG轴、RANKL/RANK/OPG与牙周炎、抑制RANKL/RANK通路治疗牙周炎的可行性等研究进展作一综述。     

抑制核因子-κB受体活化因子及其配体通路治疗牙周炎的研究进展

核因子-κB受体活化因子（RANK）及其配体（RANKL）在牙周炎患者的牙槽骨吸收中具有重要的作用,抑制RANKL／RANK通路,可有效抑制破骨细胞的分化和激活,从而抑制牙槽骨的吸收。骨保护蛋白（OPG）和RANKL结合,干扰RANKL和RANK的结合,从而防止骨组织的过度破坏。本文就RANKL／RANK／OPG轴、RANKL／RANK／OPG与牙周炎、抑制RANKL／RANK通路治疗牙周炎的可行性等研究进展作一综述。     

Effect of delayed calcium hydroxide treatment on periodontal healing in contaminated replanted teeth

The effect of delayed intracanal calcium hydroxide treatment on experimentally induced extensive inflammatory root resorption in monkeys was studied. A significant shift from inflammatory resorption to ankylosis was noted following calcium hydroxide treatment. Furthermore, calcium hydroxide treatment appeared to change the pattern of ankylosis over time, although the total ankylotic area remained the same. Ankylosis preceded by root resorption (replacement resorption) increased, while ankylosis not associated with root resorption decreased. It was concluded that intracanal calcium hydroxide treatment of teeth with compromised PDM may cause unnecessary replacement resorption if left in the root canal for a long time or changed repeatedly.     

Effect of the treatment of root surface-adhered necrotic periodontal ligament with propolis or fluoride in delayed rat tooth replantation

Objectives

The purpose of this study was to evaluate the application of 15 % propolis and 2 % acidulated-phosphate sodium fluoride solutions on the root surface-adhered necrotic cemental periodontal ligament in delayed tooth replantation.

Materials and methods

Thirty Wistar rats (Rattus norvegicus, albinus) had their right upper incisor extracted and maintained in dry storage for 60 min. After this period, the dental papilla, enamel organ, and pulp tissue were removed, and the animals were randomly assigned to three groups: group I = immersion in saline for 10 min; group II = immersion in a 2 % acidulated-phosphate sodium fluoride solution for 10 min; and group III = immersion in a 15 % propolis and propylene glycol solution for 10 min. The root canals were filled with a calcium hydroxide paste and the teeth were replanted.

Results

Inflammatory resorption, replacement resorption, and ankylosis were observed in all groups without a statistically significant difference (p?>?0.05) among them.

Conclusions

Under the tested conditions, the application of fluoride or propolis on root surface-adhered necrotic periodontal ligament did not favor the healing process in delayed tooth replantation.     

Effect of immediate calcium hydroxide treatment and permanent root-filling on periodontal healing in contaminated replanted teeth

Abstract – The effect of immediate permanent root-filling and immediate intracanal calcium hydroxide treatment on experimentally contaminated and necrotized periodontal membranes (PDM) in avulsed and subsequently replanted monkey teeth was studied. Almost the entire root surface in the non-endodontically treated control teeth were covered with inflammatory resorp-tion, A significant shift towards ankylosis not associated with root resorption (> 80% of the total root surface area) was noted following calcium hydroxide treatment. About two thirds of the root surfaces from the teeth with permanent root fillings showed surface resorptions or ankylosis preceded by root resorption. The root surface area which would have been covered by inffammatory resorption, unless endodontic treatment had been implemented, were instead predominantly covered by either surface resorption (permanent root filling) or ankylosis (calcium hydroxide treatment), Ankylosis compared with surface resorption may in the long term lead to progressive loss of root substance due to replacement resorption. It was concluded that calcium hydroxide may be an excellent initial intracanal treatment in teeth with healthy PDM, but care should be taken not to risk unnecessary development of ankylosis by prolonged treatment of teeth with compromised PDM.     

IL-1β and compressive forces lead to a significant induction of RANKL-expression in primary human cementoblasts

AIM: The aim of this study was to investigate the response of primary human cementoblasts to conditions as they occur on the pressure side during orthodontic tooth movement. METHODS: In our previous study, the cementoblasts were characterized using markers for osteoblastogenic differentiation and the cementoblast-specific marker CEMP-1. Initially, primary human cementoblasts were compressed for 1?h, 4?h, and 6?h (30?g/cm(2)). In the second experiment, the cementoblasts were stimulated with interleukin (IL)-1β for 24?h and for 96?h with 1?ng/ml and 10?ng/ml and subsequently compressed for 1?h and 6?h. Changes in mRNA expression for receptor activator of NF-κB (RANK), RANK ligand (RANKL), osteoprotegerin (OPG), and cyclooxygenase-2 (COX-2) were measured by quantitative real-time polymerase chain reaction (RT-PCR). RANK and RANKL were also examined by immunocytochemical staining at the protein level. RESULTS: Compression (30?g/cm(2)) led to a significant increase in RANKL expression after 6?h. OPG expression in compressed cementoblasts was significantly reduced after 1?h. RANK remained unchanged during the course of the experiment. Stimulation with IL-1β induced RANKL and OPG expression. However, IL-1β-dependent induction of RANKL was more prominent than the induction of OPG, leading to a (significant) increase in the RANKL/OPG ratios. The expression of RANK remained unchanged after 24?h of stimulation with IL-1β and decreased significantly after 96?h. Compression of the prestimulated cells resulted in a further increase in RANKL expression significant after 6?h. OPG and RANK expression remained unchanged compared to the unstimulated sample. COX-2 increased significantly after both compression and stimulation with IL-1β. Combined stimulation and compression resulted in a significant further increase after 6?h compared to IL-1β stimulation alone. CONCLUSION: Primary human cementoblasts in vitro express increased levels of RANKL, in particular during the combination of inflammation and compression. The increase in RANKL expression is not compensated by an increase in OPG expression. The induction of RANKL expression was associated with a significant increase in COX-2 expression. Since RANKL attracts osteoclasts, its increase might be associated with the progression of root resorption. The in vitro alterations in cementoblasts we observed may be indicators of cellular mechanisms that lead to the increased root resorption during orthodontic treatment.     

骨保护素在牙根吸收中保护作用的研究进展

［摘要］ 牙根吸收是一种生理性或病理性牙本质、牙骨质缺损的临床现象,为口腔科常见病。目前对于牙根吸收尚无有效预防手段和治疗方法。有关牙根吸收机制的研究,近几年发现骨保护素（osteoprotegerin,OPG）在此过程中起重要作用,本文就近几年OPG的结构、表达及功能,OPG/核因子kB受体活化因子配体（ligand of receptor activator of NF-κB,RANKL）/核因子kB受体活化因子（receptor activator NF-κB,RANK）系统,OPG在牙根周围相关组织中的保护作用作一综述。     

Effect of low‐level laser therapy on the healing process after tooth replantation: a histomorphometrical and immunohistochemical analysis

Abstract – Success of tooth replantation is limited because part of the replanted tooth is lost because of progressive root resorption. This study used histomorphometry and immunohistochemistry to evaluate the effect of low‐level laser therapy (LLLT) on the healing process of rat teeth replanted after different extra‐oral periods, simulating immediate and delayed replantation. Sixty Wistar rats (Rattus norvegicus albinus) had their maxillary right incisors extracted and randomly assigned to six groups (n = 10): C4, C30 and C45, in which the teeth were replanted 4 min (immediate), 30 min (delayed) and 45 min (delayed) after extraction, respectively, and L4, L30 and L45, in which the teeth were replanted after the same extra‐alveolar times, but the root surfaces and the alveolar wounds were irradiated with a gallium–aluminum–arsenate (GaAlAs) diode laser before replantation. The animals were sacrificed after 60 days. The anatomic pieces containing the replanted teeth were obtained and processed for either histomorphometrical analysis under optical microscopy or immunohistochemical expression of receptor activator of nuclear factor Kappa‐B (RANK), and its ligand (RANKL), osteoprotegerin (OPG) and tartrate‐resistant acid phosphatase (TRAP) proteins. Areas of external replacement and inflammatory root resorption were observed in all groups, without statistically significant differences (P > 0.05). Ankylosis was more frequent in L30 than in C30 (P < 0.05). RANKL immunostaining predominated over RANK and OPG immunostaining in both groups with immediate tooth replantation (P < 0.05). For the 45‐min extra‐alveolar time, however, there was greater evidence of RANK immunostaining compared to RANKL for both control and laser‐treated groups (P < 0.05). Positive TRAP immunostaining predominated in L4 and L30 (P < 0.05). In conclusion, under the tested conditions, the treatment of the root surface and the alveolar wound with LLLT did not improve the healing process after immediate and delayed tooth replantation in rats.     

Use of vitamin C in delayed tooth replantation

This study evaluated microscopically the effects of root surface treatment with three different solutions in delayed rat teeth replantation. Central incisors from 30 rats (Rattus norvegicus, albinus Wistar) were extracted and left on a bench for 6 h. The pulps were extirpated and root canals were irrigated with 1% sodium hypochlorite. After endodontic treatment, the root surfaces of all teeth were submitted to a 10-min treatment with 1% sodium hypochlorite, changed every 5 min. The teeth were then rinsed with saline for 10 min and assigned to 3 groups with ten specimens each. Groups I, II and III were treated, respectively, with 2% acidulated-phosphate sodium fluoride, vitamin C solution and effervescent vitamin C (2 g, Redoxon. After root surface treatment, the teeth were filled with calcium hydroxide and replanted. The animals were sacrificed after 10 and 60 days. Group I (fluoride) presented the largest areas of replacement resorption and ankylosis. Comparing both vitamin C groups, Group III (effervescent vitamin C) yielded better results, showing more areas of ankylosis and replacement resorption than areas of inflammatory resorption.     

Effect of immediate calcium hydroxide treatment and permanent root-filling on periodontal healing in contaminated replanted teeth

The effect of immediate permanent root-filling and immediate intracanal calcium hydroxide treatment on experimentally contaminated and necrotized periodontal membranes (PDM) in avulsed and subsequently replanted monkey teeth was studied. Almost the entire root surface in the non-endodontically treated control teeth were covered with inflammatory resorption. A significant shift towards ankylosis not associated with root resorption (greater than 80% of the total root surface area) was noted following calcium hydroxide treatment. About two thirds of the root surfaces from the teeth with permanent root fillings showed surface resorptions or ankylosis preceded by root resorption. The root surface area which would have been covered by inflammatory resorption, unless endodontic treatment had been implemented, were instead predominantly covered by either surface resorption (permanent root filling) or ankylosis (calcium hydroxide treatment). Ankylosis compared with surface resorption may in the long term lead to progressive loss of root substance due to replacement resorption. It was concluded that calcium hydroxide may be an excellent initial intracanal treatment in teeth with healthy PDM, but care should be taken not to risk unnecessary development of ankylosis by prolonged treatment of teeth with compromised PDM.     

Comparison of intracanal EndoSequence Root Repair Material and ProRoot MTA to induce pH changes in simulated root resorption defects over 4 weeks in matched pairs of human teeth

Introduction

Intracanal mineral trioxide aggregate (MTA) may provide an alternative to calcium hydroxide in the treatment of external inflammatory root resorption. This in vitro study using human matched pairs of teeth compared white ProRoot MTA (WMTA; (Dentsply Tulsa Dental Specialties, Tulsa, OK) and an alternative material with purportedly improved handling properties, EndoSequence Root Repair Material (ES; Brasseler USA, Savannah, GA), by measuring pH in simulated root surface resorptive defects after intracanal placement. The null hypothesis tested was that there is no difference between WMTA and ES.

Methods

Bilaterally matched pairs (n = 24) of extracted, human, single-rooted teeth were instrumented to apical size 50/.06, and root surface cavities were prepared at 5 mm and 2 mm from the apex. Root canals of experimental matched pairs (n = 20) were filled with WMTA or ES; control pairs (n = 4) were filled with calcium hydroxide (positive control [POS]) or saline (negative control [NEG]). Teeth were sealed coronally and apically and immersed in saline. The pH in root surface cavities was measured at 20 minutes, 3 hours, 24 hours, 1 week, 2 weeks, 3 weeks, and 4 weeks.

Results

The pH at 5 mm when compared with the 2-mm level was significantly higher for the WMTA, ES, and POS groups (P < .05, paired t tests); therefore, each level was analyzed separately. At both the 2-mm and 5-mm levels, significant pH changes occurred over time in the WMTA, ES (both P < .0001, repeated-measures analysis of variance), and POS (P < .0001, Friedman test) groups and not in the NEG group (mean pH = 7.32 ± 0.04, P > .05). There were no differences between WMTA and ES at 20 minutes and 3 hours at both levels or at 24 hours at 5mm. The pH of WMTA was higher than ES by 24 hours at the 2-mm level (8.79 vs 8.56, P < .05, paired t test) and after 1 week at the 5-mm level (8.91 vs 8.05, P < .0001) and was thereafter always significantly higher in WMTA compared with ES (P < .0001). The null hypothesis was rejected.

Conclusions

In matched pairs of teeth, intracanal placement of WMTA compared with ES resulted in a higher pH in simulated root resorption defects that was time and root level dependent.     

壳寡糖对牙周炎大鼠牙槽骨吸收及Th17/Treg平衡和OPG/RANKL/RANK通路的影响

目的: 探讨壳寡糖对牙周炎大鼠牙槽骨吸收及Th17/Treg平衡和OPG/RANKL/RANK通路的影响。方法: 建立牙周炎大鼠模型,随机分为模型组、壳寡糖低剂量组、壳寡糖中剂量组、壳寡糖高剂量组和甲硝唑组,每组12只,另取12只作为对照组。分组处理后,评估牙龈指数、牙槽骨吸收值;H-E染色观察牙周组织病理形态学变化;流式细胞术检测外周血中Th17/Treg细胞比值;酶联免疫吸附试验（ELISA）检测各组大鼠血清中IL-17、TGF-β、RANKL、OPG水平,实时荧光定量PCR（qRT-PCR）检测各组大鼠牙周组织OPG、RANKL mRNA表达水平。采用SPSS 24.0软件包对数据进行统计学分析。结果: 与对照组相比,模型组大鼠牙周组织呈现牙周膜纤维束断裂、排列紊乱,毛细血管扩张、增生,炎症细胞浸润等病理损伤;牙龈指数、牙槽骨吸收值、Th17/Treg比值、血清RANKL及IL-17水平、牙周组织RANKL mRNA水平显著升高（P<0.05）,血清OPG及TGF-β水平、牙周组织OPG mRNA水平显著降低（P<0.05）。与模型组相比,壳寡糖低、中、高剂量组和甲硝唑组大鼠牙周组织病理损伤减轻;牙龈指数、牙槽骨吸收值、Th17/Treg比值、血清RANKL及IL-17水平、牙周组织RANKL mRNA水平显著降低（P<0.05）,血清OPG及TGF-β水平、牙周组织OPG mRNA水平显著升高（P<0.05）,且壳寡糖各组呈剂量依赖性,壳寡糖高剂量组与甲硝唑组相比,差异无统计学意义（P>0.05）。结论: 壳寡糖可促使Th17/Treg平衡恢复正常,上调OPG表达,下调RANKL表达,抑制牙周炎大鼠牙槽骨吸收,改善其临床症状。     

Expression of osteoprotegerin and receptor activator of nuclear factor ��B ligand in root resorption induced by heavy force in rats

Objective

The aim of this study was to investigate the expression patterns of osteoprotegerin (OPG) and the receptor activator of nuclear factor ??B ligand (RANKL) in root resorption during orthodontic tooth movement.

Material and methods

Forty 12-week-old male SD rats were used with the right maxillary side as the experimental group and the left maxillary side as the control group. After 1?N (100g) force was loaded on the right maxillary first molar, the rats were sacrificed on days 0, 1, 4, 8, and 12. Mesial root resorption of the first molar, the number of odontoclasts and osteoclasts, and OPG and RANKL mRNA expression were determined by hematoxylin-eosin and scanning electron microscopy, tartrate-resistant acid phosphate staining, and in situ hybridization, respectively.

Results

Serious root resorption was apparent on the pressure side of the mesial root of the right maxillary first molar on days 8 and 12. The number of odontoclasts in the cementum lacuna was elevated on days 8 and 12. OPG expression rose significantly on the tensile side, while RANKL expression increased on the pressure side. The mRNA level of RANKL was significantly elevated on days 4, 8, and 12. Moreover, the RANKL/OPG mRNA ratio was increased on the pressure side, but decreased on the tensile side.

Conclusion

Changes in the expression of RANKL mRNA and the RANKL/OPG mRNA ratio are accompanied by a parallel alteration in the number of odontoclasts and tooth resorption, suggesting crucial involvement of RANKL and OPG in tooth resorption.     

乳铁蛋白对大鼠上颌扩弓及复发过程中腭中缝骨吸收的影响及机制探究

目的 研究乳铁蛋白(lactoferrin,LF)对大鼠上颌扩弓及复发过程中腭中缝破骨活动的影响,并初探其作用机制.方法 对大鼠扩弓及复发模型给予剂量为1g/kg/d的LF灌胃,用免疫组织化学染色的方法观察LF对腭中缝破骨活动的影响,检测OPG/RANKL/RANK作用轴关键因子的表达变化.结果 与单纯扩弓组相比,LF...     

Effects of calcium hydroxide addition on the physical and chemical properties of a calcium silicate-based sealer

Recently, various calcium silicate-based sealers have been introduced for use in root canal filling. The MTA Fillapex is one of these sealers, but some of its physicochemical properties are not in accordance with the ISO requirements.

Objective

The aim of this study was to evaluate the flowability, pH level and calcium release of pure MTA Fillapex (MTAF) or containing 5% (MTAF5) or 10% (MTAF10) calcium hydroxide (CH), in weight, in comparison with AH Plus sealer.

Material and Methods

The flowability test was performed according to the ISO 6876:2001 requirements. For the pH level and calcium ion release analyses, the sealers were placed individually (n=10) in plastic tubes and immersed in deionized water. After 24 hours, 7 and 14 days, the water in which each specimen had been immersed was evaluated to determine the pH level changes and calcium released. Flowability, pH level and calcium release data were analyzed statistically by the ANOVA test (α=5%).

Results

In relation to flowability: MTAF>AH Plus>MTAF5>MTAF10. In relation to the pH level, for 24 h: MTAF5=MTAF10=MTAF>AH Plus; for 7 and 14 days: MTAF5=MTAF10>MTAF>AH Plus. For the calcium release, for all periods: MTAF>MTAF5=MTAF10>AH Plus.

Conclusions

The addition of 5% CH to the MTA Fillapex (in weight) is an alternative to reduce the high flowability presented by the sealer, without interfering in its alkalization potential.