Mechanisms involved in the enhancement of osteoclast formation by enamel matrix derivative

BACKGROUND AND OBJECTIVE: Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration, and it has been reported that EMD can induce the formation of osteoclasts in mouse marrow cultures. In the present study, we investigated the mechanisms of EMD-induced osteoclast formation using a mouse monocytic cell line, RAW 264.7. MATERIAL AND METHODS: Bioactive fractions were purified from EMD by reverse-phase HPLC using a C18 hydrophobic support, following which RAW 264.7 cells were cultured with EMD or its purified fractions in the presence of receptor activator of nuclear factor-kappaB ligand (RANKL) for 8 d. Following staining with tartrate-resistant acid phosphatase (TRAP), TRAP-positive multinucleated cells were counted. The expression of receptor activator of nuclear factor-kappaB (RANK), as well as phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase, in RAW 264.7 cells were detected using immunoblotting. To determine whether EMD has an effect on osteoclast function, differentiated RAW 264.7 cells were cultured on Osteologic Multitest slides with RANKL in the presence of EMD. RESULTS: Purified EMD fractions (fraction numbers 21-25; EMD peak 2) were found to enhance the formation and function of RAW 264.7 cells induced by RANKL. Moreover, EMD peak 2 enhanced the levels of phosphorylation of ERK p38 and RANK in RAW 264.7 cells stimulated with RANKL. CONCLUSION: Our results indicate that EMD induces the formation of osteoclasts through interaction with RANKL, while ERK and p38 MAPK may play a critical role in the enhancement of osteoclast formation in RAW 264.7 cells.     

釉基质蛋白对人牙龈上皮细胞增殖的影响

目的:研究釉基质蛋白(enamelmatrixproteins,EMPs)对体外培养的人牙龈上皮细胞增殖活性的影响。方法:乙酸提取法获取EMPs,取龈切术中切除的牙龈组织,采用酶消化法培养牙龈上皮细胞。将第1代牙龈上皮细胞按照22500个/孔接种,分为4组:对照组(不加EMPs)及EMPs分别为50、100、200μg/ml的实验组。采用MTT法检测各组细胞数量,对每个时间点的各组实验数据进行方差分析。结果:人牙龈上皮细胞能在EMPs覆盖的平皿上生长。统计学分析表明,不同浓度的EMPs在早期对牙龈上皮细胞的增殖无显著影响;从第3天开始,当培养液中EMPs浓度为200μg/ml时,牙龈上皮细胞增殖显著受抑。结论:EMPs影响牙龈上皮细胞的增殖,并存在剂量和时间依赖性。     

Enamel matrix derivative alone or in combination with a bioactive glass in wide intrabony defects

This controlled clinical study investigated the clinical and radiographic outcome of wide intrabony periodontal defects treated by enamel matrix derivatives alone or in combination with a bioactive glass over a period of 8 months. Twenty-three chronic periodontitis patients, who received initial therapy and had radiographical interproximal defects with an associated probing depth of 6 mm or more and an intrabony component of at least 4 mm, were included. Each of the patients, contributing at least one intrabony defect, was treated with either enamel matrix derivative alone (group 1, n=10) or the combination (group 2, n=13). In both groups, all clinical and radiographical parameters were improved. Groups 1 and 2 presented a mean pocket reduction of 5.03±0.89 and 5.73±0.80 mm, recession of 0.97±0.24 and 0.56±0.18 mm, relative attachment gain of 4.06±1.06 and 5.17±0.85 mm, and radiographic bone gain of 2.15±0.42 and 2.76±0.69 mm, respectively. An intergroup comparison revealed significant differences for all of the parameters, yielding a more favorable outcome towards the combined approach. Within the limits of the study, both treatments resulted in marked clinical and radiographical improvements, but combined treatment seemed to enhance the results in the treatment of wide intrabony defects.     

牙本质基质蛋白1基因反义核酸对MDPC-23细胞碱性磷酸酶活性和矿化能力的影响

目的 :观察牙本质基质蛋白 1(dentalmatrixprotein ,DMP1)基因反义寡核苷酸 (AS -ODN)对成牙本质细胞系MDPC -2 3细胞碱性磷酸酶 (ALPase)活性和矿化能力的影响 ,深入研究成牙本质细胞的分化和牙本质形成的机制。方法 :以稳定表达DMP1的MDPC -2 3细胞为靶细胞、不同浓度DMP1反义核酸为阻断剂 ,观察不同作用时间对MDPC -2 3细胞ALPase活性的影响。用Westernblot法检测 10 μmol/LAS -ODN不同作用时间细胞DMP1表达情况 ,并观察连续作用 10d对该细胞矿化能力的影响。结果 :与正常组和S -ODN组比较 ,不同浓度的AS -ODN能够降低MDPC -2 3细胞ALPase活性 ,其中 10 μmol/LAS -ODN降低ALPase活性最为显著 (作用 5d时OD值最低 ,为 0 .3 3 78± 2 .0E)。Westernblot法检测到DMP1在MDPC -2 3细胞的表达在 10 μmol/LAS -ODN加入 12h后减弱 ,2 4h后完全阻断。此浓度AS -ODN连续作用细胞 10d后 ,vonKossa染色显示细胞中出现明显的钙盐沉积 ,矿化结节的数量和大小明显少于对照组。结论 :DMP1反义核酸能够降低MDPC -2 3细胞AL Pase活性和矿化能力 ,提示DMP1参与调节成牙本质细胞矿化过程 ,可能具有启动牙本质矿化的作用。     

脱细胞异体真皮在预防腮腺手术后Frey综合征中的作用

目的：评价脱细胞异体真皮用于预防腮腺术后味觉出汗综合征的疗效。方法：2004年1月～2005年4月间住院手术治疗的腮腺良性肿瘤患者168例，其中多形性腺瘤89例，腺淋巴瘤45例，基底细胞腺瘤17例，其他17例。根据患者意愿分为2组，第1组104例，施行腮腺浅叶或部分切除术；第2组64例，在腮腺浅叶或部分切除术后，根据缺损大小，于腮腺咬肌筋膜瓣和剩余腮腺组织之间植入脱细胞异体真皮。所有病例均通过调查问卷形式对味觉出汗综合征进行评价，并随机抽取60例（2组各30例）进行两侧面部碘一淀粉试验（starchiodinetest）。应用SPSS10．0统计软件包对所得数据进行X^2检验。结果：问卷调查显示，第1组63／104例（60．58％）、第2组1／64例（1．56％）出现味觉出汗综合征主观症状。客观检查的患者中，第1、2组Frey综合征客观症状的发生例数分别是24例和2例。术后第1组18例、第2组1例出现涎漏。2组检验结果，X^2=32．851，P＜0．05，有显著差异。结论：脱细胞异体真皮植入是预防腮腺区手术后发生味觉出汗综合征的有效方法。     

Ten-year results following treatment of intra-bony defects with enamel matrix proteins and guided tissue regeneration

Background: Surgery utilizing an enamel matrix protein derivative (EMD) or guided tissue regeneration (GTR) has been shown to promote periodontal regeneration.
Aim: To evaluate the 10-year results following treatment with EMD, GTR, EMD+GTR, and open flap debridement (OFD).
Material and Methods: Thirty-eight patients out of an initial group of 56 participants were treated with one of the four modalities. Results were evaluated before surgery, at 1 year, and at 10 years. Primary outcome variable was CAL change.
Results: Treatment with EMD yielded a mean CAL gain of 3.4±1.0 mm ( p <0.001) and 2.9±1.4 mm ( p <0.001) at 1 and 10 years, respectively. GTR resulted in a mean CAL gain of 3.2±1.4 ( p <0.001) at 1 year and 2.8±1.2 mm ( p <0.001) at 10 years. Mean CAL gain in the EMD+GTR group was of 3.3±1.1 mm ( p <0.001) and 2.9±1.2 mm ( p <0.001) at 1 and 10 years, respectively. Treatment with OFD demonstrated a mean CAL gain of 2.0±1.2 mm ( p <0.01) at 1 year and 1.8±1.1 mm ( p <0.01) at 10 years. Compared with OFD, the three regenerative treatments resulted in statistically significant ( p <0.05) higher CAL gain, at both 1 and 10 years. The CAL change between 1 and 10 years did not present statistically significant differences in any of the four groups.
Conclusion: The present results indicate that the clinical outcomes obtained with all four approaches can be maintained over a period of 10 years.     

Periodontal tissue engineering and regeneration: Consensus Report of the Sixth European Workshop on Periodontology

Aim: To review the scientific preclinical background and clinical studies of current methods of periodontal regeneration in the treatment of infrabony defects and soft tissue deficiencies
Method: Five commissioned review papers including two systematic reviews were scrutinized by a group of experts in order to derive consensus conclusions, clinical relevance/implications and to propose future research requirements.
Results: The following five papers were assessed:
  1. Biological mediators and periodontal regeneration: a review of enamel matrix proteins at the cellular and molecular levels.
  2. Regeneration of periodontal tissues: combination of barrier membranes and grafting materials – Biological foundation and preclinical evidence.
  3. Clinical outcomes with bioactive agents alone or in combination with grafting or GTR
  4. Treatment of gingival recession with coronally advanced flap procedures. A systematic review.
  5. Soft tissue management at implant sites     

Inhibition of human embryonic palatal mesenchymal cell cycle by secalonic acid D: a probable mechanism of its cleft palate induction

OBJECTIVE: To assess the mechanism(s) of cleft palate induction by secalonic acid D (SAD) in human embryonic palatal mesenchymal (HEPM) cells and compare them with those evaluated in the murine embryonic palate. DESIGN: Effect of SAD on HEPM cell proliferation was studied by obtaining dose response curves for cell numbers, uptake of 3H-thymidine and the expression of proliferating cell nuclear antigen (PCNA). Effects of SAD on cell cycle were assessed by flowcytometry. Cell-labeling with 3H-glucosamine and immunoblot analysis were conducted to study SAD effects on the synthesis of glycosaminogycans (GAG) and the expression of fibronectin and tenascin, respectively. RESULTS: SAD induced a concentration-dependent decrease in HEPM cell number and 3H-thymidine uptake beginning at 0.1 microg of SAD/ml. Expression of PCNA and progression of cell cycle from G1 to S phase were inhibited following SAD exposure. Cell viability was significantly reduced only at 7.5 microg/ml of SAD or higher indicating that the reduction in cell numbers by SAD at lower concentrations is likely due to reduced proliferation and at higher concentrations due to both reduced proliferation and cell death. Synthesis of extra cellular matrix components (GAGs, fibronectin or tenascin) by HEPM cells, however, was not inhibited by SAD. CONCLUSION: The results of these studies confirmed those of our previous studies with mice and the MEPM cells that SAD may induce cleft palate by reducing numbers of palatal mesenchymal cells by inhibition of their proliferation thereby leading to a reduction in the size of the developing palate shelves.     

Langerhans cells express matrix metalloproteinases 9 and 2 and tissue inhibitors of metalloproteinases 1 and 2 in healthy human gingival tissue and in periodontitis

BACKGROUND: As antigen-presenting cells, Langerhans cells may play an important role in the initiation and maintenance of periodontal disease. This study is the first report that extends our knowledge of the expression of matrix metalloproteinases and their endogenous tissue inhibitors by Langerhans cells in healthy and diseased gingival tissues. METHODS: Single and double immunolabeling procedures were carried out using monoclonal antibodies against CD1a, matrix metalloproteinases 2 and 9, and tissue inhibitors of matrix metalloproteinases 1 and 2, and analyzed by conventional and confocal microscopes. RESULTS: Langerhans cells expressed matrix metalloproteinases 2 and 9, and tissue inhibitors of matrix metalloproteinases 1 and 2 in healthy and diseased gingival tissues. The tissue inhibitors of matrix metalloproteinase-positive Langerhans cells were mainly observed in the upper epithelial layers. Matrix metalloproteinase 9-positive Langerhans cells were observed especially during periodontitis and in the basal epithelial layer or crossing the basement membrane. CONCLUSION: During periodontal disease, changes in the expression of matrix metalloproteinases and their tissue inhibitors by gingival Langerhans cells could be implicated in the migration of the cells towards the connective tissue.