牙髓干细胞对牙周炎中破骨细胞的作用

目的研究牙髓干细胞（DPSC）对牙周炎中破骨细胞形成及牙槽骨再生的影响,并初步探索DPSC对小鼠破骨细胞的作用机制。 方法体外诱导小鼠骨髓单核细胞破骨分化,观察破骨细胞组（OC组）及其与DPSC共培养组（OC+DPSC组）的抗酒石酸酸性磷酸酶（TRAP）染色情况,实时荧光定量聚合酶链反应（PCR）检测破骨分化相关基因包括活化T细胞核因子（NFATc1）、基质金属蛋白酶9（MMP-9）及TRAP的表达差异。体内构建小鼠慢性牙周炎模型,通过微计算机体层摄影（micro-CT）扫描后三维重建,比较慢性牙周炎+0.9%氯化钠溶液注射组（NS组）和慢性牙周炎+DPSC注射组（DPSC组）釉牙骨质界至牙槽嵴顶（CEJ-ABC）距离,并对标本进行苏木精-伊红和TRAP染色,观察DPSC对小鼠破骨细胞及牙槽骨再生的影响。采用SPSS 20.0软件进行数据统计分析,采用独立样本t检验及校正t检验分析组间差异。 结果体外TRAP染色发现,与DPSC共培养明显抑制成熟破骨细胞形成,OC+DPSC组成熟破骨细胞均数（4.2 ± 0.2）少于OC组均数（6.8 ± 0.2）,差异有统计学意义（t= 15.922,P<0.001）;破骨细胞表面积均数（0.046 ± 0.007）mm²也明显小于OC组（0.763 ± 0.015）mm²,差异有统计学意义（t = 83.174,P<0.001）。相对OC组,OC+DPSC共培养组的MMP-9、NFATc1及TRAP的mRNA相对表达量明显降低,均值分别为0.38 ± 0.17（t = 6.217,P = 0.003）、0.24 ± 0.12（t = 10.569,P = 0.003）和0.55 ± 0.13（t = 6.077,P = 0.026）。micro-CT扫描结果显示,DPSC注射组CEJ-ABC的平均距离为（0.215 ± 0.017）mm,明显小于0.9%氯化钠溶液组（0.311 ± 0.022）mm,差异有统计学意义（t= 10.921,P<0.001）,组织学观察下DPSC组炎症反应较0.9%氯化钠溶液组轻,且破骨细胞更少。 结论DPSC可通过抑制牙周炎破骨细胞的形成从而促进牙槽骨再生,有望作为一种可局部注射的骨代谢双向调节生物制剂,治疗临床上包括牙周炎等因骨代谢失衡引起的炎症性骨吸收疾病。     

唑来膦酸对破骨细胞黏附及整合素αv、β3基因表达的影响

目的 研究唑来膦酸（ZOL）对破骨细胞黏附以及整合素α_v和β₃基因表达的影响。方法 体外诱导小鼠RAW264.7细胞向破骨细胞分化,通过抗酒石酸酸性磷酸酶（TRAP）染色及牙本质吸收陷窝检测以评价破骨细胞生成情况。将细胞分为对照组及ZOL处理组两组,后者用1×10^-6 mol·L^-1的ZOL处理2 d,用结晶紫染色法检测细胞黏附情况,用实时荧光定量聚合酶链反应、Western blot和免疫荧光化学法检测整合素α^v、β³ mRNA及蛋白表达水平。结果 TRAP染色及牙本质吸收陷窝检测提示有多核破骨细胞生成。ZOL处理组破骨细胞黏附能力较对照组显著降低（P<0.01）。ZOL处理组整合素α_v、β₃mRNA水平分别为0.66±0.05、0.59±0.08,显著低于对照组的1.01±0.01和1.01±0.02（P<0.01）;蛋白表达水平分别为31 934.84±112.91、18 812.79±194.13, 较对照组（52 517.81±211.72、31 441.93±456.87）分别下降了39.19%和40.17%（P<0.01）。免疫荧光化学检测显示,ZOL处理使整合素α_v、β₃荧光强度（9.491±0.748、4.744±0.759）较对照组（15.159±1.143、11.418±1.095）分别降低了37.39% 和58.45%（P<0.01）。结论 ZOL可抑制破骨细胞黏附并下调整合素α_v、β₃表达;ZOL的上述作用可能参与对破骨细胞性骨吸收的抑制。     

Effects of whole cell sonicates of Treponema lecithinolyticum on osteoclast differentiation

BACKGROUND: Alveolar bone destruction is a characteristic feature of periodontal diseases and multinucleated osteoclast cells derived from hemopoietic cells are responsible for bone resorption. Treponema lecithinolyticum is a novel oral spirochete isolated from the periodontal lesions. METHODS: The effect of whole cell sonicates on the osteoclast differentiation was examined in a co-culture system of hemopoietic mouse bone marrow cells and calvaria derived-osteoblastic cells to clarify the role of T. lecithinolyticum in the alveolar bone destruction associated with periodontal diseases. The differentiated osteoclasts were confirmed by tartrate-resistant acid phosphatase (TRAP) staining. RESULTS: Sonicates of this bacterium stimulated the osteoclast formation in the co-culture system in a dose-dependent manner. The sonicates-induced osteoclast formation was partially inhibited by the heat treatment of sonicates. Indomethacin, which is a prostaglandin inhibitor, decreased the osteoclast formation induced by the bacterial sonicates. CONCLUSIONS: These findings suggest that T. lecithinolyticum induces osteoclast differentiation by a prostaglandin E2-dependent mechanism and that heat-labile components may be involved in this process.     

牙周膜成纤维细胞对外周血单个核细胞分化的影响

目的：探讨牙周膜成纤维细胞在破骨细胞形成过程中作用;观察破骨样细胞的生长过程。方法：本实验以含有1α,25（OH）2D3和地塞米松的培养基将牙周膜成纤维细胞、单个核细胞分别进行单独或直接共培养,每3d对TRAP阳性多核破骨细胞的数量及牙本质磨片的吸收陷窝数目和面积分别进行记录、计算。结果：不同时间段间的TRAP阳性单个核细胞与TRAP阳性多核细胞数目相比较,差异具有统计学意义（P〈0.05）;同时,不同组间的吸收陷窝数目和面积比较,差异具有显著性（P〈0.001）。牙周膜成纤维细胞明显增加了共培养组TRAP阳性多核细胞数量、吸收陷窝数目和面积。然而牙周膜成纤维细胞组与单个核细胞组之间的吸收陷窝数目与吸收陷窝面积差异无统计学意义。结论：末梢血单个核细胞需在牙周膜成纤维细胞存在的条件下,才能形成多核的破骨样细胞。同时,在共培养中,可以发现破骨样细胞在体外存活的时间短暂。     

主穹隆蛋白通过抑制破骨细胞分化在牙周炎中起骨保护作用

目的:通过建立小鼠实验性牙周炎模型及体外骨髓间充质干细胞(BMMSCs)破骨细胞向诱导,探讨主穹隆蛋白(MVP)在牙周炎骨吸收中的作用。方法:MVP基因敲除(MVP-/-)和野生型(WT)C57BL/6小鼠分别局部注射脂多糖(LPS)以建立实验性牙周炎模型,通过micro CT扫描、耐酒石酸酸性磷酸酶(TRAP)染色等方法检测骨吸收程度。同时,体外分离培养MVP-/-与WT C57BL/6小鼠的BMMSCs,并诱导其向破骨细胞分化,通过TRAP染色、麦胚凝集素(WGA)染色等方法观察MVP对BMMSCs破骨向分化及骨吸收活性的影响。结果:在LPS诱导的小鼠实验性牙周炎中,MVP-/-组小鼠牙周炎骨吸收更为明显,且在注射区域内可见更多破骨细胞;体外实验证明,MVP-/-组小鼠的BMMSCs分化形成更多的破骨细胞,且骨吸收更明显。结论:MVP可以抑制破骨细胞分化,在牙周炎中起骨保护作用。     

破骨细胞分化因子诱导小鼠骨髓细胞形成破骨细胞的实验研究

目的:探讨破骨细胞分化因子(ODF)和巨噬细胞集落刺激因子(M-CSF)联合应用进行体外诱导小鼠骨髓细胞形成破骨细胞(OC)的能力。方法:收集5～6周小鼠骨髓细胞,在含M-CSF(10 ng/ml)的α-MEM全培养液中培养24h,然后将悬浮生长的细胞接种到24孔培养板,并加入不同浓度的ODF和M-CSF。,通过观察抗酒石酸盐酸性磷酸酶(TRAP)染色的阳性细胞和能否在骨磨片上形成吸收陷窝来鉴定OC形成情况。结果:在只加入ODF或M-CSF一种细胞因子时,未见有TRAP阳性或CTR阳性细胞形成,同时加入ODF和M-CSF,可形成典型的OC。TRAP阳性多核细胞的数目和培养液中钙离子浓度的增加与ODF的浓度呈正相关。结论:小鼠骨髓来源的单核细胞在ODF和M-CSF共同作用下可形成具有骨吸收功能的OC,为体外研究OC的分化发育和功能调节提供了一种新的方法。     

Autocrine regulation of osteoclast formation and bone resorption by IL-1 alpha and TNF alpha.

Bone resorption is regulated by the cytokines within marrow cells that mediate osteoclast formation and activation. IL-1 and TNF induce bone resorption by stimulating the production of osteoclast-like multinucleated cells and by increasing the bone-resorbing activity of formed osteoclasts. This study was designed to detect IL-1 and TNF in osteoclasts in vitro and to determine whether these cytokines up-regulate osteoclast differentiation and bone resorption. The production of IL-1 alpha, -beta, and TNF alpha, beta in osteoclasts was examined immunohistochemically and by in situ hybridization. In the co-culture of C57BL/6N mouse bone marrow and MC3T3-G2/PA6 cells, a colony of osteoclasts formed, and IL-1 alpha and TNF alpha were detected. However, IL-1 beta and TNF beta were not detected. To investigate the role of IL-1 alpha and TNF alpha from osteoclasts, we enumerated TRAP-positive cells and measured the resorption pit areas in the presence of antibodies against IL-1 alpha and TNF alpha. The addition of antibodies against IL-1 alpha and TNF alpha to the co-culture system decreased the number of TRAP-positive colonies at seven days after incubation (anti-IL-1 alpha, 25.0 +/- 2.3%; anti-TNF alpha, 41.7 +/- 3.7%; anti-IL-1 alpha + anti-TNF alpha, 40.5 +/- 1.3%; and control, 100%), and the ratio of mononuclear to multinuclear cells had changed (anti-IL-1 alpha, 90:10; anti-TNF alpha, 75:25; anti-IL-1 alpha+ anti-TNF alpha, 88:12; and control, 60:40). The total pit areas per dentin slice also decreased with the addition of antibodies (anti-IL-1 alpha, 28,828; anti-TNF alpha, 49,249; anti-IL-1 alpha + anti-TNF alpha, 30,685; and control, 303,139 mm2). These results suggest that local production of IL-1 alpha and TNF alpha by osteoclasts is an important mechanism for regulating the osteoclast differentiation and bone resorptive process.     

白细胞介素-6对破骨细胞骨吸收效应及MMP-3表达影响的实验研究

目的：观察不同浓度IL－6对破骨细胞骨吸收的剂量效应及对破骨细胞基质金属蛋白酶－3表达的影响，以期进一步阐明IL－6介导基质金属蛋白酶在破骨细胞性骨吸收中的病理机制。方法：采用体外破骨细胞溶骨模型，通过原子吸收分光光度仪及免疫组化染色技术检测不同浓度IL－6对破骨细胞溶骨活性及其质金属蛋白酶－3表达的影响。结果：当IL－6＞10U／ml时，培养上清中Ca^2 浓度显著增加，牙本质片上骨吸收陷窝数目明显增多；当IL－6浓度为100U／ml、500U／ml时破骨细胞基质金属蛋白酶－3表达的阳性信号显著增强。结论：在IL－6作用下，破骨细胞表达基质金属蛋白酶－3。IL－6对破骨细胞具有激活作用，低浓度主要诱导破骨细胞形成，较高浓度刺激破骨细胞的溶骨活性。     

Magnesium vs. machined surfaced titanium - osteoblast and osteoclast differentiation

PURPOSE

This study focused on in vitro cell differentiation and surface characteristics in a magnesium coated titanium surface implanted on using a plasma ion source.

MATERIALS AND METHODS

40 commercially made pure titanium discs were prepared to produce Ti oxide machined surface (M) and Mg-incorporated Ti oxide machined surface (MM). Surface properties were analyzed using a scanning electron microscopy (SEM). On each surface, alkaline phosphatase (ALP) activity, alizarin red S staining for mineralization of MC3T3-E1 cells, and quantitative analysis of osteoblastic gene expression, were evaluated. Actin ring formation assay and gene expression analysis of TRAP and GAPDH performing RT-PCR were performed to characterize osteoclast differentiation on mouse bone marrow-derived macrophages (BMMs).

RESULTS

MM showed similar surface morphology and surface roughness with M, but was slightly smoother after ion implantation at the micron scale. M was more hydrophobic than MM. No significant difference between surfaces on ALP activity at 7 and 14 days were observed. Real-time PCR analyses showed similar levels of mRNA expression of the osteoblast phenotype genes; osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), and collagen 1 (Col 1) in cell grown on MM at 7, 14 and 21 days. Alizarin red S staining at 21 days showed no significant difference. BMMs differentiation increased in M and MM. Actin ring formation assay and gene expression analysis of TRAP showed osteoclast differentiation to be more active on MM.

CONCLUSION

Both M and MM have a good effect on osteoblastic cell differentiation, but MM may speed the bone remodeling process by activating on osteoclast differentiation.     

缺氧与加力在正畸骨吸收作用中的细胞学研究

目的 观察缺氧对正畸压力侧破骨细胞形成的影响,探讨缺氧、加力在正畸骨改建中各自所起的作用,并分析其在破骨细胞形成过程中的相互作用关系.方法 建立人牙周膜细胞与外周血单个核细胞接触式共培养模型,模拟正畸缺氧、加力、缺氧+加力,不同条件作用1、3、6h后培养3d,TRAP染色检测破骨样细胞的形成.Reahime PCR检测骨改建相关因子HIF-1α 、IL-1β、RANKL、OPGmRNA的表达并计算RANKL/OPG比值.结果 TRAP染色阳性细胞在缺氧+加力组各个处理时间段都有出现,缺氧组则在处理3、6h中出现,加力组仅在处理6h中出现,且缺氧+加力组的阳性细胞数目均大于单独缺氧、加力组.HIF-1α、IL-1β、RANKL、OPGmRNA的表达及RANKL/OPG比值都呈现出缺氧+加力组均大于单纯缺氧、加力组.结论 缺氧和加力分别可以诱导与人牙周膜细胞共培养的单个核细胞向破骨细胞分化,缺氧和加力相互协同、共同促进正畸牙齿移动过程中骨吸收的发生.     

Interferon-gamma down-regulates gene expression of cathepsin K in osteoclasts and inhibits osteoclast formation

The cytokine, IFN-gamma, has been shown in vitro to inhibit bone resorption, but the mechanisms responsible for this inhibition have not been clearly defined. Cathepsin K is a major protease responsible for bone resorption. IFN-gamma may inhibit bone resorption through down-regulation of osteoclast genes, including cathepsin K. To test the hypothesis, we investigated the effect of IFN-gamma on cathepsin K expression in the MOCP-5 and wild-type mouse bone marrow co-culture systems by Northern blot as well as osteoclast formation at different stages of differentiation. The results show that IFN-gamma down-regulates mRNA levels of cathepsin K in a time- and dose-dependent manner. Consequently, cathepsin K protein production is also reduced by IFN-gamma. Moreover, our results indicate that IFN-gamma inhibits osteoclast formation only early in osteoclast differentiation. IL-6 and TNFalpha did not significantly affect cathepsin K gene expression in osteoclasts. However, IL-1alpha stimulated gene expression. In conclusion, our data suggest that the actions of IFN-gamma on osteoclastic bone resorption may be mediated by its effects on both osteoclast formation at an early stage and osteoclast gene expression in mature osteoclasts.     

Mineral trioxide aggregate inhibits osteoclastic bone resorption

Mineral trioxide aggregate (MTA), a commonly used endodontic repair material, is useful for both basic and clinical research, and the effect of MTA on osteoblast differentiation has been well-defined. However, the effects of MTA on osteoclastic bone resorption are not fully understood. Hence, the aim of this study is to examine the effect of MTA solution in the regulation of osteoclast bone-resorbing activity using osteoclasts formed in co-cultures of primary osteoblasts and bone marrow cells. MTA solution dose-dependently reduced the total area of pits formed by osteoclasts. The reduction of resorption induced by 20% MTA treatment was due to inhibition of osteoclastic bone-resorbing activity and had no effect on osteoclast number. A 20% MTA solution disrupted actin ring formation, a marker of osteoclastic bone resorption, by reducing phosphorylation and kinase activity of c-Src, and mRNA expressions of cathepsin K and mmp-9. A high concentration of MTA solution (50%) induced apoptosis of osteoclasts by increasing the expression of Bim, a member of the BH3-only (Bcl-2 homology) family of pro-apoptotic proteins. Taken together, our results suggest that MTA is a useful retrofilling material for several clinical situations because it both stimulates osteoblast differentiation and inhibits bone resorption.     

Effects of 1,25 - （OH） 2 D3 on Stimulation of Osteoclast - like Cells Formationand Expression of ODF mRNA in Murine Marrow Cells in Vitro

目的：观察不同浓度的1，25-(OH)2D3对破骨细胞形成及对骨髓细胞ODFmRNA表达的影响：进一步阐明骨吸收刺激因子在正畸牙周组织改建中的作用。方法：应用不同浓度的1，25-(OH)2D3(0、10^-10、10^-8、10^-6mol／L)诱导大鼠骨髓细胞破骨样细胞的形成，采用体外破骨细胞溶骨模型，观察牙本质片上骨吸收陷窝数目，采用原位杂交技术检测骨髓基质细胞ODF的mRNA表达。结果：随着1，25-(OH)2D3浓度的增加，骨陷窝数明显增多，陷窝面积增大；ODF mRNA表达的阳性信号显著增强。结论：在体外，1，25-(OH)2D3可以调节破骨细胞的骨吸收活性，进而调节局部骨微环境的骨吸收及骨形成平衡的变化。     

Avagacestat抑制破骨细胞形成及溶骨功能改善骨关节炎骨破坏

目的 探究γ-分泌酶抑制剂Avagacestat通过抑制破骨细胞的形成及溶骨功能,抑制骨关节炎的进展。方法 提取6周龄C57鼠骨髓细胞诱导为巨噬细胞扩增培养,加入M-CSF及RANKL诱导后通过CCK8测定Avagacestat对该细胞的半数抑制浓度(IC₅₀),设置0、120、240 nmol/L 3种Avagacestat浓度组,对比破骨细胞形成实验、功能实验并检测3组细胞TRAP、C-fos、Cath-K等破骨相关基因的表达。建立LPS诱导关节炎实验动物模型,通过三维骨重建检测软骨下骨溶解情况、通过HE染色及TRAP染色检测破骨细胞分布情况。结果 在24、48、96 h 3个时间点,Avagacestat对破骨细胞的IC₅₀分别为493、426、405 nmol/L。TRAP实验显示Avagacestat抑制破骨细胞形成,骨板骨吸收实验证实Avagacest抑制溶骨功能,且240 nmol/L浓度的Avagacestat对破骨细胞形成及功能的抑制显著高于120 nmol/L;RT-PCR结果提示,加药后溶骨相关基因TRAP、C-fos、...     

白介素-6与1,25(OH)_2维生素D_3联合作用下大鼠骨髓破骨细胞的体外诱导培养

目的观察联合应用白介素(IL)-6和1,25(OH)2D3对大鼠骨髓破骨细胞生成诱导的影响。方法采用4周龄SD大鼠无菌条件下取出股骨,剪去两骺端,以α-MEM培养液将骨髓细胞冲出,然后将细胞悬液接种于预置盖玻片或牙本质片的24孔培养板内,24 h后换液。试验分为4组,A组:不加任何诱导因子;B组:加入IL-6(10 U/ml);C组:加入1,25(OH)2D3(1×10-8mol/L);D组:加入IL-6(10 U/ml)和1,25(OH)2D3(1×10-8mol/L)。每组各6孔,于培养的第7天进行多核细胞计数。结果培养1周左右IL-6与1,25(OH)2D3在体外均可单独诱导骨髓破骨细胞的形成,但D组多核细胞数与B、C组相比差异有显著性(P<0.05)。结论IL-6与1,25(OH)2D3联合作用下对骨髓破骨细胞生成的诱导效果明显高于单因素诱导效果。     

Facilitation of bone resorption activities in synovial lavage fluid patients with mandibular condyle fractures

The aim of this study was to investigate the bone resorption effect of the mediators delivered in joint cavity of patients with mandibular condyle fractures by detecting osteoclast markers using cellular biochemistry methods, and by analysing bone resorption activities via inducing osteoclast differentiation of the infiltrated cells from arthrocentesis. Sixteen joints in 10 patients with mandibular condyle fractures were evaluated. The control group consisted of synovial fluid (SF) samples from seven joints of four volunteers who had no clinical signs or symptoms involving the temporomandibular joint (TMJ) or disc displacement. We collected SF cells from all patients during therapeutic arthrocentesis. The infiltrating cells from TMJ SF were cultured, differentiated into tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclast‐like cells and examined bone resorption activities. We also investigated factors related to osteoclast induction of SF, using ELISA procedures. Osteoclast‐like cells were induced from the SF cells obtained from all patients with condylar fractures. These multinucleated giant cells were positive for TRAP and actin, and had the ability to absorb dentin slices. The levels of macrophage colony‐stimulating factor (M‐CSF), prostaglandin E2 (PGE2), soluble form of receptor activator of nuclear factor kappa‐B ligand (sRANKL) and osteoprotegerin (OPG), in SF samples from the patients, were significantly higher than in the controls. These findings indicate that bone resorption activities in SF from patients with mandibular condyle fractures were upregulated and may participate in the pathogenesis and wound healing.     

Effects of vascular endothelial growth factor-C and -D on osteoclast differentiation and function in human peripheral blood mononuclear cells

ObjectiveThe purpose of this study was to clarify the interaction of vascular endothelial growth factors (VEGFs)-C and -D with cell surface foetal liver kinase-1 (Flk-1) and fms-like tyrosine kinase-4 (Flt-4) receptors in the induction and activity of osteoclasts in cultured human peripheral blood mononuclear cells (PBMCs).DesignPBMCs were cultured on chamber slides or on ivory discs for 2 or 3 weeks in the presence of macrophage-colony stimulating factor (M-CSF), VEGF-A, -C or -D, or placental growth factor (PlGF) with or without receptor activator of nuclear factor kappa-B ligand (RANKL). The number of osteoclasts in each group was counted and the area of ivory resorption was measured. In addition, osteoclast differentiation was further analysed under the same conditions, but with the addition of specific neutralizing antibodies against Flk-1 and Flt-4.ResultsRANKL was essential for the induction of osteoclasts in PBMCs. However, significant differences were found in the number of osteoclasts induced by VEGF-A, -C, -D or M-CSF with RANKL compared with control groups lacking or containing RANKL. Blocking of either Flk-1 or Flt-4 resulted in a reduction in the enhancement of osteoclast differentiation in PBMCs by VEGF-C or -D with RANKL. The osteoclasts induced by VEGF-A, -C, -D or M-CSF with RANKL formed significantly larger resorption lacunae than those formed by osteoclasts induced by RANKL alone.ConclusionsThis study showed that VEGF-C and -D play a role in the induction of osteoclast differentiation through both Flk-1 and Flt-4 receptors and influence the area of the ivory resorption in PBMCs.     

Identification of RANKL in osteolytic lesions of the facial skeleton

RANKL (receptor activator of nuclear factor kappaB ligand) promotes osteoclast differentiation, stimulates osteoclast activity, and prolongs osteoclast survival and adherence to bone. Abnormalities of the RANKL/RANK/osteoprotegerin system have been implicated in a range of diseases, including osteoporosis. To date, no work has been done in osteolytic lesions of the facial skeleton. In this study, specimens of ameloblastomas, dentigerous cysts, odontogenic keratocysts, and radicular cysts were subjected to immunohistochemical analysis for RANKL and tartrate-resistant acid phosphatase (TRAP). Immunofluorescence staining for TRAP was visualized under confocal microscopy. All specimens demonstrated distinct positive immunoreactivity to RANKL and TRAP. The TRAP-positive cells also stained with in situ hybridization for human calcitonin receptor, a definitive marker for osteoclasts. Mononuclear pre-osteoclasts were observed to migrate from blood to the connective tissue stroma and multinucleate toward the bone surface. It can be concluded that RANKL plays a role in bone resorption in osteolytic lesions of the facial skeleton.     

Inhibition of osteoclastogenesis by the secretion of osteoprotegerin in vitro by rat dental follicle cells and its implications for tooth eruption

Tooth eruption requires the presence of the dental follicle, a loose connective tissue sac that surrounds each unerupted tooth. Early postnatally in the rat, the follicle secretes colony-stimulating factor-1 (CSF-1) and monocyte chemotactic protein-1 (MCP-1), chemotactic molecules that are probably responsible for the recruitment of mononuclear cells. These cells, in turn, fuse to form osteoclasts, which are required for alveolar bone resorption to form an eruption pathway. Recent studies have shown that the osteoprotegerin (OPG) gene is expressed in the dental follicle, but in the first mandibular molar of the rat, that expression is reduced at day 3, the time of maximal osteoclast numbers on the alveolar bone. Inhibition of OPG expression at this time would allow osteoclast formation/activation. To determine if the dental follicle cells do secrete OPG that inhibits osteoclastogenesis, spleen cell cultures were established and soluble osteoclast differentiation factor (ODF) and CSF-1 added to some of them to promote osteoclast formation. In other cultures, dental follicle cells were added in an insert, such that they did not touch the spleen cells. Using a quantitative, tartrate-resistant acid phosphatase (TRAP) assay, it was shown that ODF and CSF-1 promoted osteoclastogenesis in the spleen cell cultures, but the addition of the follicle cells inhibited this and returned the TRAP activities to those seen in cultures of spleen cells only. Adding anti-OPG to these cultures, however, negated the effect of the follicle cells, demonstrating that OPG was the inhibitory molecule secreted by those cells. The follicle cells also immunostained for OPG, confirming that they synthesize OPG. These findings, coupled with those of other studies which show that the periodontal ligament (a derivative of the dental follicle) also secretes OPG, indicate that, except for the period of time in tooth eruption, where osteoclast formation is needed to form an eruption pathway, secretion of OPG would be the norm, presumably to prevent resorption of alveolar bone and subsequent disruption of the periodontal ligament.