Growth of oral and skin fibroblasts from patients with oral submucous fibrosis

The purpose of the investigation was to evaluate and compare the proliferation (growth) of mouth fibroblasts and skin fibroblasts from patients with oral submucous fibrosis (OSF). Material comprised fibroblasts from fibrous bands situated in the buccal mucosa and from the inner aspect of the forearm of 8 patients with classic features of OSF as well as fibroblasts from 6 buccal mucosa and 8 skin biopsy specimens from healthy non-areca nut chewing individuals. Cells were cultured for 8 days according to standard techniques. Their growth was monitored daily, under optimal conditions as well as exposure to concentrations of arecoline. The data were analyzed using regression analysis, analysis of variance and the Kruskal-Wallis test. We found no statistically significant differences between the proliferation patterns of oral and skin fibroblasts from patients or between those from patients and controls. The reaction of the cells exposed to concentrations of arecoline was similar; at low concentrations (0.1–10 μg/ml) normal growth was maintained, while 100 μg/ml inhibited growth. It is concluded that fibroblasts from mouths affected by OSF have proliferation patterns which fall within normal parameters, that the excessive collagen formation in established OSF is not due to increased fibroblast proliferation and that arecoline does not stimulate fibroblast proliferation.     

IL-1β干预后人牙周膜成纤维细胞中TRAF6的基因表达研究

目的：研究IL-1β干预后人牙周膜成纤维细胞中TRAF6的基因表达水平的变化，为细胞因子网络调控牙周膜成纤维细胞功能的研究提供新资料。方法：采用原代培养的人牙周膜成纤维细胞，取5-8代细胞以IL-1β梯度浓度干预，采用RT-PCR法检测TRAF6在人牙周膜成纤维细胞中的表达。结果：在正常的人牙周膜成纤维细胞中未见TRAF6基因表达，IL-1β干预后，TRAF6呈阳性表达，并且随着IL-1β干预的浓度增高，其表达水平上调。结论：IL-1β干预后TRAF6在HPLFs中的表达水平的变化提示TRAF6可能是参与牙周膜成纤维细胞功能调控的重要信号分子。     

Biologic interaction of three-dimensional periodontal fibroblast spheroids with collagen-based and synthetic membranes

Background: Cell‐based therapy using autologous cells has been suggested as a potential approach for periodontal tissue regeneration. Spheroid systems are a form of three‐dimensional cell culture that promotes cell matrix interaction, which could recapitulate the aspect of cell homeostasis in vivo. The aim of this study is to assess the interaction of periodontal fibroblast spheroids with synthetic and collagen‐based membranes that have been used in guided tissue regeneration. Methods: Commercially available normal human periodontal ligament fibroblasts were grown in spheroid forms using a liquid overlay technique and then transplanted onto a collagen‐based and a polyglycolic acid–based membrane. The biologic interaction of the spheroids with the membranes was assessed using basic histology, Alamar blue tissue viability assay, scanning electron microscopy, and immunohistochemical analysis. Results: Periodontal fibroblast spheroids adhered to both membranes, and the cells were able to proliferate and migrate from the spheroids both horizontally and vertically into the membrane scaffolds. Immunohistochemical analysis showed expression of collagen type I, periostin, and Runx2 by the periodontal fibroblasts. Conclusion: Periodontal fibroblast spheroids were able to grow three‐dimensionally on the biologic membranes and may have the potential to be used together with guided tissue regeneration approaches as an adjunct for periodontal regeneration.     

Nicotine-induced damage affects gingival fibroblasts in the gingival tissue of rats

Background: Very limited information is available from in vivo studies about whether smoking and/or nicotine affect gingival tissues in the absence of plaque. The purpose of this study is to evaluate the effect of the systemic administration of nicotine in the proliferation and counting of fibroblast‐like cells in the gingival tissue of rats. Methods: Thirty adult male Wistar rats were randomly assigned into two groups to receive subcutaneous injections of a saline solution (control group = group C) or nicotine solution (group N; 3 mg/kg) twice a day. The animals were euthanized 37, 44, or 51 days after the first subcutaneous injection. Specimens were routinely processed for serial histologic sections. Five fields of view in the connective tissue adjacent to the gingival epithelium and above the alveolar bone crest of the maxillary first molar were selected for the counting of fibroblast‐like cells. Data were statistically analyzed (P <0.05). Results: The intergroup analysis detected a lower number of fibroblast‐like cells in group N compared to group C on days 37 (2.65 ± 1.41 and 6.67 ± 3.25, respectively), 44 (2.70 ± 1.84 and 8.57 ± 2.37, respectively), and 51 (2.09 ± 1.41 and 7.49 ± 2.60, respectively) (P <0.05). The quantification of fibroblast‐like cells showed no significant difference (P >0.05) in the intragroup analysis of control and nicotine throughout experimental periods. In the intergroup analysis, group N had reduced proliferating cell nuclear antigen–positive fibroblasts compared to group C in all periods (P <0.05). Conclusion: The daily systemic administration of nicotine negatively affected, in vivo, the number and proliferation of fibroblast‐like cells in the gingival tissue of rats.     

Matrix metalloproteinase-2 expression induced by two different adhesive systems on human pulp fibroblasts

Introduction

This study evaluated the expression of matrix metalloproteinase-2 (MMP-2) in primary cultures of human pulp fibroblasts (HPFs) when exposed to extracts from dentin-bonding systems.

Methods

Polymerized resin disks of the bonding agent of a 2-step self-etch adhesive (TechBond, Isasan, Rovello Porro, Italy) or of the primer/bonding agent a 2-step etch-and-rinse adhesive (Optibond Solo; Sybron-Kerr, Orange, CA) were immersed in HPF culture medium for 24 or 96 hours. HPFs were incubated in the adhesive-conditioned or control (untreated) culture medium for 24 hours. Western blot and immunofluorescence analyses were performed to assay MMP-2 expression.

Results

MMP-2 expression levels in HPFs cultured for 24 hours in culture medium were similar in both the control and experimental media groups showing a faint band at 67 kDa. Conversely, the HPFs incubated in the medium that contain polymerized resin disks for 96 hours showed increased MMP-2 expression compared with the untreated medium. The self-etch adhesive displayed the most pronounced induction of MMP-2 expression. These findings were confirmed by immunofluorescence analysis.

Conclusions

HPFs display increased MMP-2 expression after 96 hours of conditioning of the HPF culture medium with polymerized disks of dentin bonding systems. This MMP-2 expression/activation may represent a defence mechanism exhibited by HPFs towards monomers eluted from the dentin bonding systems.     

周期性牵张力加载下人牙周膜细胞中血管内皮生长因子表达变化的研究

目的：观察周期性牵张力作用下，体外培养的人牙周膜成纤维细胞(human periodontal ligament fibroblasts，HPLFs)中血管内皮生长因子(Vascular endothelial growth factor，VEGF)表达的变化，以探讨机械力介导下牙周组织改建的机制。方法：通过体外细胞培养加载系统，采用周期性牵张力(每分钟6个循环，12％形变量)加载l、3、5、7d，用免疫组化和原位杂交技术，观察VEGF在HPLFs中的表达。结果：HPLFs在体外表达VEGF，其蛋白水平及mRNA水平的表达从加力ld开始升高，5d达到高峰，在加力7d开始下降。结论：在一定条件下，周期性牵张力可显著影响HPLFs VEGF的蛋白和mRNA水平的表达，进一步证实机械力作用下，VEGF参与了牙周组织改建，在正畸牙齿移动的机制中具有重要意义。     

金栀含漱液对人牙周膜成纤维细胞毒性的实验研究

目的：采用MTT法评价中药制剂金栀含漱液、氢氧化钙药尖及FC对人牙周膜成纤维细胞（HPDLF）的细胞毒性及毒性的可逆性。方法：体外培养HPDLF，选用金栀含漱液作为实验组（A组），氢氧化钙药尖浸提液为阴性对照组（B组），FC（C组）为阳性对照组，培养基作为空白对照组，分别与HPDLF接触1、3、5、7d时用Mr丌法测细胞增殖率及接触第1天的回复度。结果：A组及B组对HPDLF增殖无显著性抑制作用，细胞毒性可逆；C组对HPDLF增殖有显著的抑制作用，细胞毒性不可逆。FC组与金栀含漱液组和氢氧化钙药尖组相比抑制作用均有显著性差异，后两组的作用与培养基相当。结论：中药制剂金栀含漱液对HPDLF无明显细胞毒性作用。