Donor variability in the proliferation of human dental pulp fibroblasts

Fibroblasts were isolated from human dental pulps of healthy third molars from 49 donors of ages ranging from 17 to 68. Significant variability was noted in the success of obtaining primary cultures from these pulps. Variability between the various cultures was also observed in the reliability of maintaining subcultures of the primary cultures as well as recovery from frozen stocks of established cell lines. Of the original 49 explant cultures studied, only three survived long-term passage and freezing. In addition to difficulties and variability in establishing cell lines, the human pulp fibroblasts also showed great variability in proliferative activity which could not be accounted for by donor age, source, or passage number. These findings highlight significant difficulties in establishing reliable human pulp fibroblast cultures and the need for great care in interpreting any in vitro data.     

转录因子Klf4在人牙髓组织和牙髓成纤维细胞中的表达研究

目的研究转录因子Klf4在人牙髓组织和体外培养的牙髓成纤维细胞中的表达特点。方法采用免疫荧光技术检测Klf4和增殖标记物ki67在人牙髓组织及体外培养的牙髓成纤维细胞中的表达情况;利用逆转录聚合酶链反应(RT-PCR)检测Klf4在人牙髓细胞和矿化诱导14 d的表达情况。结果人牙髓中,Klf4主要表达在成牙本质样细胞和血管内皮细胞中;在体外培养的牙髓成纤维细胞中,与诱导前相比,Klf4在矿化诱导14 d时的表达明显增强。结论 Klf4在成牙本质细胞中呈特异性表达,可能与成牙本质细胞分化相关。     

血管内皮生长因子在人牙髓中的表达

目的：研究血管内皮生长因子（ vascular endothelial growth factor VEGF） 在正常、深龋或炎症牙髓中的表达及其意义。方法：采用免疫组化SABC法，对牙髓中的VEGF的表达进行组织学定位，并通过Image pro-plus 5.1图像分析软件对成牙本质细胞和牙髓成纤维细胞中VEGF染色进行平均光密度值（optical density OD）测定。利用SPSS13．0统计软件对各组数据进行单因素方差分析或秩和检验。结果：人牙髓中，VEGF主要表达在血管内皮细胞、成牙本质样细胞和牙髓成纤维细胞的胞浆中。正常组成牙本质细胞的VEGF表达较其它两组弱（P〈0.01）。与正常组相比，牙髓成纤维细胞中VEGF的表达在深龋组明显增强（P〈0．05），而在炎症组明显减弱（P〈0．05）。此外，VEGF在炎症组的某些炎细胞如中性粒细胞、浆细胞的胞浆中也有表达。结论：VEGF在龋病、牙髓炎中的变化可能与牙髓炎的发生、炎症发展有关，并且可能参与了成牙本质样细胞对牙髓损伤的修复过程。     

Interleukin-1beta activity and collagen synthesis in human dental pulp fibroblasts

Immunopathologic reactions play a significant role in inflammatory diseases of dental pulp. Interleukin-1beta (IL-1beta) is recognized as a key player in mediating cellular immune response. In this study, we measured the content of IL-1beta and its effect on collagen synthesis in cultures of fibroblasts derived from healthy and diseased dental pulps. We found that diseased pulp fibroblasts contain 2.5-fold greater amounts of IL-1beta and synthesized 80% greater amounts of collagen compared with healthy pulp fibroblasts. However, exogenous IL-1beta failed to stimulate collagen synthesis by diseased fibroblasts, whereas collagen synthesis by healthy pulp fibroblasts was stimulated by more than 2-fold. These observations imply that pulp disease induces abnormalities associated with fibroblast response toward IL-1beta.     

牙髓成纤维细胞的免疫相关作用研究进展

成纤维细胞作为牙髓的主要细胞,其基本功能是合成胶原和基质.近年的研究表明成纤维细胞在牙髓免疫炎症反应中发挥着重要作用.本文从成纤维细胞产生炎症介质、合成生长因子等几个方面对其免疫相关作用作一综述.     

血小板衍化生长因子对人牙髓成纤维细胞DNA和胶原蛋白合成的影响

目的:观察血小板衍化生长因子(platelet-derived growth factor,PDGF)对人牙髓成纤维细胞(human dental pulp fibroblast,HDPF )DNA合成和胶原蛋白合成的影响.方法:应用3H-TdR和3H-脯氨酸掺入方法,观察PDGF对体外培养的HDPF DNA合成和胶原蛋白合成的情况.结果:20～60 ng/mL PDGF可明显促进HDPF DNA的合成,40 ng/mL浓度使细胞DNA合成在36 h达最大值;对细胞的胶原蛋白合成无明显促进作用.结论:PDGF明显促进HDPF DNA的合成,可能在治疗牙髓病中起重要作用.     

血小板衍化牛长因子对人牙髓成纤维细胞DNA和胶原蛋白合成的影响

目的：观察血小板衍化生长因子（platelet-derived growth factor,PDGF）对人牙髓成纤维细胞（human dental pulp fibroblast,HDPF）DNA合成和胶原蛋白合成的影响。方法：应用^3H-TdR和^3H-脯氨酸掺入方法，观察PDGF对体外培养的HDPF DNA合成和胶原蛋白合成的情况。结果：20-60ng/mL PDGF可明显促进HDPF DNA的合成，40ng/mL浓度使细胞DNA合成在36h达最大值；对细胞的胶原蛋白合成无明显促进作用。结论：PDGF明显促进HDPF DNA的合成，可能在治疗牙髓病中起重要作用。     

Stimulatory effect of laser irradiation on calcified nodule formation in human dental pulp fibroblasts

The purpose of the present study was to investigate the effect of laser irradiation on calcified nodule formation in human dental pulp (HDP) cells. HDP cells were irradiated once with a Ga-Al-As laser for 5 and 10 min, and calcified nodule formation was determined by von Kossa staining. The laser irradiation increased the number of calcified nodules in a time-dependent manner. The activity of alkaline phosphatase and production of collagen and osteocalcin in conditioned medium were measured. Both were higher in the irradiated group than in the nonirradiated group. These results suggested that formation of calcified nodules in HDP cells, as well as in alkaline phosphatase activity, the production of collagen and osteocalcin were enhanced by laser irradiation.     

Epidermal growth factor released in human dental pulp following orthodontic force

This study investigated the role of human epidermal growth factor (EGF) in the angiogenic response of the dental pulp to orthodontic force. The release of angiogenic growth factor EGF in human dental pulp following orthodontic force application was examined using neutralizing antibody anti-human (anti-h) EGF to block its effects. The dental pulps from 10 premolar teeth from 10 patients (equal numbers of males and females aged 11-14 years), treated with a straightwire fixed appliance for 2 weeks and extracted for orthodontic reasons, were divided vertically, and sections from each half-pulp were individually co-cultured with a section of rat aorta in collagen surrounded by growth media. Anti-h EGF was added to the media of the co-cultures from one-half of each pulp from each tooth from each patient; the remaining co-cultures from the other half of each pulp without anti-h EGF were used as the controls. Cultures were examined daily by light microscopy for angiogenic growth and number of microvessels. The addition of anti-h EGF to the growth media in the co-cultures resulted in a significant reduction (P < 0.05, Wilcoxon signed rank test) in pulpal and rat aorta microvessel numbers, compared with the control co-cultures. The results indicate that EGF released following orthodontic force application plays a part in the angiogenic response of the pulp.     

Lymphangiogenesis in human dental pulp

AIM: To investigate the impact of inflammation on lymphangiogenesis in human dental pulp. METHODOLOGY: Eleven samples of dental pulp without inflammation and 11 dental pulps with moderate to intense mononuclear cell inflammatory infiltrate associated with dentine caries were selected. The streptavidin-biotin complex stain was used to detect CD31, vascular endothelial growth factor receptor-3 (VEGFR-3) and alpha-smooth muscle actin. The number of lymphatic vessels was obtained by counting the number of vessels positive for CD31 and VEGFR-3 and negative for alpha-smooth muscle actin. RESULTS: The results demonstrated that the mean number (+/-SD) of vessels positive for CD31 and VEGFR-3 (lymphatic vessels) in the group with inflammation (6.09 +/- 1.81) was statistically higher (P = 0.0123) than the mean number in the group without inflammation (3.73 +/- 2.20). CONCLUSION: Increased co-immunostaining of CD31 and VEGF-3 in vessels associated with human dental pulp inflammation occurred, which suggests lymphangiogenesis.     

Role of human pulp fibroblasts in angiogenesis

After pulp amputation, complete pulp healing requires not only reparative dentin production but also fibroblast proliferation, nerve fiber growth, and neoangiogenesis. This study was designed to investigate the role of pulp fibroblasts in angiogenesis. Human pulp fibroblasts from third molars co-cultured with human umbilical vein endothelial cells induced the organization of endothelial cells and the formation of tubular structures corresponding to capillaries in vivo. The direct contact between both cells was not necessary to induce angiogenesis, and the observed effect was due to soluble factors. This was confirmed with neutralizing antibodies against FGF-2 and VEGF, which decreased the angiogenic effects of these soluble factors. Immunohistochemistry showed that both FGF-2 and VEGF were expressed in human dental pulp fibroblasts, and this expression increased after injury. These results suggest that the pulp fibroblasts secrete angiogenic factors, which are necessary for complete pulp healing, particularly at the pulp injury site.     

Biosynthesis of glycoproteins by rabbit dental pulp fibroblasts in culture

Confluent dental pulp fibroblast cultures incorporated l-[³H]-fucose in a linear manner with time into non-diffusible macromolecules over 24 h. Ascorbate supplementation did not appear to alter the amount or type of macromolecules. Equilibrium CsCl-density-gradient centrifugation established that the [³H]-fucose-labelled macromolecules released into the medium were predominantly glycoproteins. Sodium dodecyl sulphate-polyacrylamide-gel electrophoresis showed that the major fucosylated glycoprotein had an apparent molecular weight of 230,000, but several other species were also seen. The major fucosylated glycoprotein was fibronectin by its affinity for gelatin and its immunoprecipitation with a specific anti-(cold-insoluble globulin).     

人类β-防御素在正常、深龋和炎症牙髓中的表达

目的:研究人类β-防御素(human beta-defensins,HBD)在正常、深龋和炎症牙髓中的表达及其意义。方法:采用免疫组化SP法分别对正常、深龋及炎症牙髓中β-防御素HBD-1、HBD-4的表达进行组织学定位,并通过Image pro-plus 5.1图像分析软件对HBD-1/HBD-4染色进行平均光密度值的测定。所得数据用SPSS 13.0统计软件进行相应的统计学分析。结果:在人牙髓中,β-防御素主要表达在成牙本质细胞的细胞核及细胞突起中。HBD-1在3组内表达无显著差异(P>0.05);HBD-4在深龋和牙髓炎组中的表达与正常组相比均显著增强(P<0.05),且炎症组亦强于深龋组,差异有统计学意义(P<0.05)。结论:HBD-1参与牙髓天然免疫系统;HBD-4参与龋病发展过程中牙髓组织的免疫防御反应。     

4种牙本质粘结剂对牙髓成纤维细胞毒性作用的比较

目的:对比研究4种第七代牙本质粘结剂对人牙髓成纤维细胞的毒性作用,初步探讨其生物安全性。方法:组织块培养法原代培养人牙髓成纤维细胞,免疫组织化学染色SP法鉴定细胞来源,采用四甲基偶氮唑盐比色法和浸提液法,双盲观察4种新一代牙本质粘结剂(G-Bond、i-Bond、xeno V、Clearfil S3Bond)的不同体积分数浸提液(12.5%、25%、50%、100%)作用不同时间(24、48、72 h)对人牙髓成纤维细胞的毒性作用。结果:4种牙本质粘结剂不同体积分数浸提液的作用下,人牙髓成纤维细胞形态均发生不同程度的变化。24、48 h时,xeno V细胞毒性影响较小,i-Bond细胞毒性影响较大,G-Bond与Clearfil S3 Bond细胞毒性无明显差异;72 h时,4种牙本质粘结剂对细胞毒性影响无明显差异。结论:4种牙本质粘结剂毒性趋于0～2级,随着作用时间延长,细胞毒性无明显差异。