MyD88 expression in the rat dental follicle: implications for osteoclastogenesis and tooth eruption

Liu D, Yao S, Wise GE. MyD88 expression in the rat dental follicle: implications for osteoclastogenesis and tooth eruption. Eur J Oral Sci 2010; 118: 333–341. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci Myeloid differentiation factor 88 (MyD88) is a key adaptor molecule in the interleukin (IL)‐1 and IL‐18 toll‐like receptor signaling pathways. Because MyD88 is present in dental follicle (DF) cells in vitro, the purpose of this study was to determine its chronological expression in vivo, as well as its possible role in osteoclastogenesis and tooth eruption. An oligo DNA microarray was used to determine expression of the Myd88 gene in vivo in the DFs from the first mandibular molars of postnatal rats from days 1 to 11. The results showed that MyD88 was expressed maximally on day 3. Using small interfering RNA (siRNA) to knock down MyD88 expression in the DF cells also reduced the expression of the nuclear factor‐kappa B‐1 (NFKB1) and monocyte chemoattractant protein 1 (MCP‐1) genes. Interleukin‐1α up‐regulated the expression of NFKB1, MCP‐1, and receptor activator of nuclear factor kappa B ligand (RANKL), but knockdown of MyD88 nullified this IL‐1α effect. Conditioned medium from DF cells with MyD88 knocked down had reduced chemotactic activity for mononuclear cells and reduced osteoclastogenesis, as opposed to controls. In conclusion, the maximal expression of MyD88 in the DF of postnatal day 3 rats may contribute to the major burst of osteoclastogenesis needed for eruption by up‐regulating MCP‐1 and RANKL expression.     

Expression of endothelial monocyte-activating polypeptide II in the rat dental follicle and its potential role in tooth eruption

Endothelial monocyte-activating polypeptide II (EMAP-II) is an inflammatory cytokine with chemotactic activity. Because the dental follicle (DF) recruits mononuclear cells (osteoclast precursors) to promote the osteoclastogenesis needed for tooth eruption, it was the aim of this study to determine if EMAP-II contributes to this recruitment. Using a DNA microarray, EMAP-II was found to be highly expressed in vivo in the DFs of day 1 to day 11 postnatal rats, with its expression elevated on days 1 and 3. Use of a short interfering RNA (siRNA) to knock down EMAP-II expression resulted in a reduction in the expression of colony-stimulating factor-1 ( CSF-1) and monocyte chemoattractant protein-1 ( MCP-1) in the DF cells. Addition of EMAP-II protein to the DF cells partially restored the expression of CSF-1 and MCP-1. In chemotaxis assays using either conditioned medium of the DF cells with anti-(EMAP-II) immunoglobulin G added or conditioned medium of DF cells with EMAP-II knocked down by siRNA, migration indexes of bone marrow mononuclear cells were significantly reduced. These results suggest that EMAP-II is another chemotactic molecule in the dental follicle involved in the recruitment of mononuclear cells, and that EMAP-II may exert its chemotactic function directly by recruiting mononuclear cells and indirectly by enhancing the expression of other chemotactic molecules (CSF-1 and MCP-1).     

表皮生长因子在牙齿萌出通道形成中的作用研究

目的:研究表皮生长因子(EGF)在牙齿萌出过程中,是否参与软、硬组织通道的形成。方法:①免疫组化法检测表皮生长因子及其受体在出生后13、15 d以及成年小鼠下颌第一磨牙萌出部位口腔黏膜的表达变化;②原代培养Wistar大鼠牙囊细胞,选择生长良好的第3代细胞,MTT法筛选EGF作用于牙囊细胞的较佳效应浓度。将第3代牙囊细胞以1×105/孔接种到培养皿中,加入最佳浓度的EGF孵育0.5、1、3、6 h后,Trizol一步法分别提取总RNA,采用反转录聚合酶链反应(RT-PCR)检测同一浓度的EGF作用不同时间后,牙囊细胞MCP-1 mRNA的表达变化。结果:①牙萌出时,EGF在萌出牙齿冠方黏膜的上皮层呈弱阳性表达,表皮生长因子受体(EGFR)在口腔上皮全层呈强阳性表达。而牙萌出后,EGF的表达集中于口腔黏膜的固有层,EGFR的表达集中于上皮基底层;②在EGF浓度为5～10 ng/mL时,对牙囊细胞的增殖具有明显促进作用(P<0.05),其中10 ng/mL促进作用最强。牙囊细胞与10 ng/mL的EGF共同孵育0.5、1、3、6 h均能明显促进牙囊细胞MCP-1 mRNA的表达(P<0.05),其中3 h时促进作用最强,以后逐渐恢复,但仍比对照组高(P<0.05)。结论:EGF及其受体可能促进萌出牙齿冠方实性上皮团的形成;适当浓度的EGF能显著增加牙囊细胞的增殖活性,并上调牙囊细胞中MCP-1 mRNA的表达。     

Recombinant human bone morphogenetic protein-2 for peri-implant bone regeneration: a case report

Background: Currently, clinicians have a limited treatment arsenal in the repair of peri‐implant defects. The aim of the present report is to present the clinical results of treating a dental implant using recombinant human bone morphogenetic protein (rhBMP)‐2 in an elderly patient. Methods: A 75‐year‐old man presented for routine dental prophylaxis. Clinical and radiographic examination revealed significant loss of attachment and bone loss around an implant replacing the maxillary left first molar. The patient did not report any symptoms, and the implant showed no signs of mobility. Because of the severity of the defect, regenerative treatment using a combination of rhBMP‐2 and freeze‐dried bone allograft was used. Results: The patient was followed for 80 weeks postoperatively. By 28 weeks, significant probing depth reduction and radiographic bone fill was observed, and the original implant crown was replaced. From 28 weeks postoperatively to 80 weeks, no significant clinical or radiographic changes were observed. Conclusions: rhBMP‐2 represents a potential therapeutic modality for severe peri‐implant hard tissue loss. Future studies should examine parameters, such as surgical technique, to maximize the rhBMP‐2‐driven regenerative outcomes.     

Chronology and regulation of gene expression of RANKL in the rat dental follicle

Tooth eruption in the rat requires bone resorption resulting from a major burst of osteoclastogenesis on postnatal day 3 and a minor burst of osteoclastogenesis on postnatal day 10 in the alveolar bone of the first mandibular molar. The dental follicle regulates the major burst on postnatal day 3 by down-regulating its osteoprotegerin (OPG) gene expression to enable osteoclastogenesis to occur. To determine the role of receptor activator of nuclear factor-kappa B ligand (RANKL) in tooth eruption, its gene expression was measured on postnatal days 1-11 in the dental follicle. The results show that RANKL expression was significantly elevated on postnatal days 9-11 in comparison to low expression levels at earlier time-points. As OPG expression is high at this latter time-point, this increase in RANKL expression would be needed for stimulating the minor burst of osteoclastogenesis. Tumor necrosis factor-alpha enhances RANKL gene expression in vitro and it may be responsible for up-regulating RANKL in vivo. Transforming growth factor-beta1 and interleukin-1alpha also enhance RANKL gene expression in vitro but probably have no effect in vivo because they are maximally expressed early. Bone morphogenetic protein-2 acts to down-regulate RANKL expression in vitro and, in vivo, may promote alveolar bone growth in the basal region of the tooth.     

RNAi的作用机制及其在口腔医学领域的研究进展

RNA干扰是由双链RNA诱发的使特定序列基因沉默的现象,存在小干扰RNA(siRNA)和微小RNA(miRNA)两种不同的途径,主要有转录水平基因沉默,转录后水平基因沉默和翻译水平基因沉默三种机制,具有高效性和高特异性的特点。本文就RNAi的途径、技术路线及RNA干扰在牙齿发育、唇腭裂研究、牙周组织和口腔肿瘤中的研究进展作一综述。     

白细胞介素-10对人牙囊细胞OPG表达的影响

目的：研究人牙囊细胞白细胞介素-10（IL-10）蛋白的表达及其对骨保护素（08teoprotegerin，OPG）表达的影响。方法：采用组织块加胶原酶消化法培养人牙囊细胞，进行IL-10免疫组化染色。将25ng／ml IL-10作用于牙囊细胞0h、1h,3h,6h,9h，用ELISA法检测上清液中OPG含量变化。结果：人牙囊细胞IL-10表达阳性。25ng／ml IL-10作用于人牙囊细胞3-6hOPG表达显著增加（P〈0．05）。结论：人牙囊细胞表达IL-10，IL-10通过增加人牙囊细胞OPG表达，在牙齿萌出过程中起重要的调控作用。     

siRNA抑制人Tca8113/CDDP细胞多药耐药相关蛋白表达的研究

目的:利用小干扰RNA(small interfering RNA,siRNA)抑制人舌癌耐药细胞多药耐药相关蛋白(muhidrugresistance associated protein,MRP)的表达,观察其对培养细胞耐药性的干扰情况。方法:设计针对MRP基因的siRNA,转染人舌癌多药耐药细胞Tca8113/CDDP,用RT-PCR分析MRP mRNA的表达;免疫细胞化学技术检测MRP蛋白表达量;MTT法检测MRP siRNA的细胞毒作用。结果:Tca8113/CDDP细胞转染MRP siRNA后,MRPmRNA表达较对照组明显降低,降低率为61.3%;转染36 h后MRP siRNA组的MRP蛋白表达阳性率较对照组明显减低;MRP siRNA组中顺铂对Tca8113/CDDP细胞生长的抑制作用明显强于对照组,且抑制作用随时间的延长而增强。结论:MRP siRNA可以明显抑制口腔癌耐药细胞的多药耐药。     

Tissue engineering with recombinant human bone morphogenetic protein-2 for alveolar augmentation and oral implant osseointegration: experimental observations and clinical perspectives

Surgical placement of oral implants is governed by the prosthetic design and by the morphology and quality of the alveolar bone. Nevertheless implant placement often appears difficult, if at all possible, due to aberrations of the alveolar ridge. Hence prosthetically dictated implant positioning often entails augmentation of the alveolar ridge and adjoining structures. In this review we discuss recent observations of the biologic potential, clinical relevance, and perspectives of application of recombinant human bone morphogenetic protein-2 (rhBMP-2) technologies for alveolar bone augmentation and oral implant osseointegration. Using discriminating critical-size supraalveolar defects and clinical modeling in dogs, we show that rhBMP-2 has a substantial potential for augmenting alveolar bone and supporting osseointegration of titanium oral implants. Moreover, using clinical modeling, we demonstrate re-osseointegration in advanced periimplantitis defects and long-term functional loading of titanium oral implants placed into rhBMP-2-induced bone. Our studies suggest that inclusion of rhBMP-2 for alveolar bone augmentation and oral implant fixation will not only enhance the predictability of existing clinical protocol but also allow new approaches to these procedures.     

VEGFsiRNA对人舌癌Tca8113细胞移植瘤血管生成影响的研究

目的:观察小干扰RNA(small interfering RNA,siRNA)抑制血管内皮生长因子(vascular endothelial growth factor,VEGF)表达对裸鼠皮下人舌癌Tca8113细胞移植瘤血管生成的影响.方法:构建人舌癌Tca8113细胞20只裸鼠皮下移植瘤模型,随机分为4组.将两对针对VEGF的siRNA真核表达载体(Pu-VEGF-siRNA1,Pu-VEGF-siRNA2)脂质体法作瘤体内及瘤周注射;以注射空质粒组作实验对照组;未注射组作空白对照组.注射1次/3 d×10次,末次注射3d后,剥离瘤体,RT-PCR检测瘤组织VEGF-mRNA表达,免疫印迹技术检测瘤组织VEGF蛋白表达.免疫组化染色法观察VEGF阳性染色及微血管参数.结果:Pu-VEGF-siRNA2组移植瘤VEGF-mRNA表达及VEGF蛋白表达、总体微血管密度、有腔微血管密度、有腔血管平均血管周长及管腔面积均较空质粒组及空白对照组低(p＜0.05).而Pu-VEGF-siRNA1组未观察到上述差别(P＞0.05).结论:siRNA能在体内抑制舌癌VEGF表达,有效减少肿瘤血管生成.不同的干扰片段体内作用效果存在差异.     

人骨形成蛋白－2对人牙髓细胞增殖和分化的影响

目的：探讨骨形成蛋白－2（BMP－2）对体外培养的人牙髓细胞（DPCs）增殖和分化的影响。方法：体外培养人DPCs,分别与不同浓度的BMP－2（50、100、200 ng／mL）共同培养;分别检测各组细胞的增殖情况、碱性磷酸酶（alkaline phosphatase,ALP）活性、钙化结节形成量以及牙本质涎磷蛋白（DSPP）、牙本质基质蛋白－1（DMP－1）各成牙本质相关基因的表达水平。结果：50～200 ng／mL 的BMP－2对DPCs的增殖均无明显促进作用;但是,能呈剂量依赖性地提高细胞的ALP活性、促进钙化结节的形成、上调DSPP和DMP－1 mRNA的表达水平,各浓度BMP－2组均高于对照组（P ＜0．05）。结论：BMP－2对体外培养的人DPCs增殖无明显影响,但可明显促进DPCs的成牙本质细胞方向分化。     

提取并部分纯化bBMP促进节段性骨缺损愈合的定量组织学研究

目的　从小牛骨基质中提取并部分纯化bBMP ,以促进节段性骨缺损的愈合。方法　通过SDS PAGE电泳及生物活性鉴定实验 ,证实bBMP保留了其活性蛋白成分 ,具有良好的骨诱导活性。在此基础上 ,合成一种新型复合人工骨材料即HA bBMP Co ,用以整复家兔尺骨中段 1.5cm节段性骨缺损 ,并对缺损区新骨生长量进行定量组织学测定。结果　HA bBMP Co复合人工骨植入整复节段性骨缺损 ,其新骨生长量明显高于同期HA Co复合人工骨组 (P <0 .0 1)。结论　HA bBMP Co复合人工骨能有效地促进节段性骨缺损的愈合 ,是一种具有良好应用前景的骨替代材料