釉基质蛋白对猪骨髓基质细胞增殖和根面附着生长的影响

目的:研究釉基质蛋白(enamel matrix proteins,EMPs)对体外培养的猪骨髓基质细胞(bone marrowstrom alcells,BMSCs)增殖和在根面附着生长的影响,为EMPs联合应用BMSCs修复牙周组织缺损提供理论依据。方法:抽取猪髂骨骨髓,全血培养法获得骨髓基质细胞。培养液中EMPs的浓度分别为25、50、100、200μg/ml,以不加EMPs为对照。MTT法测定各组细胞的增殖活性。对实验数据行单因素方差分析和SNK法组间比较,检验水准为α=0.05。制备猪自体牙根片,以200μg/mlEMPs处理组为实验组,对照组不用EMPs处理。接种BMSCs后培养7d,HE染色和扫描电镜观察。选取800倍下标准视野,计数每个视野中的细胞数,取4个视野均值。采用配对t检验法作统计学分析,检验水准为α=0.05。结果:猪BMSCs在含有EMPs的培养液中生长良好。EMPs对BMSCs的促增殖作用呈浓度和时间依赖性,200μg/ml浓度的EMPs从实验的第3天开始显著促进猪BMSCs增殖。HE染色显示BMSCs在根片表面附着良好。扫描电镜观察表明实验组附着生长的BMSCs数量较多,与对照组相比具有显著差异。结论:EMPs在200μg/ml浓度时可显著促进猪BMSCs增殖,并促进其在根面的附着生长,提示EMPs和BMSCs可以联合应用以修复牙周组织缺损。     

釉基质蛋白对猪骨髓基质细胞黏附、伸展及增殖的影响

目的：研究釉基质蛋白（enamel matrix proteins,EMPs）对体外培养的猪骨髓基质细胞（bone marrow stromal cells,BMSCs）黏附、伸展和增殖活性的影响。方法：抽取猪髂骨骨髓．全血培养法获得骨髓基质细胞。培养液中EMPs的浓度分别为25、50、100、200μg／ml,以不加EMPs为对照。用比色法检测不同浓度EMPs对BMSCs黏附的影响。通过计数预定视野中伸展的细胞数,计算BMSCs在培养1h、3．5h、6、5h后的伸展率。MTT法测定各组细胞的增殖活性。对实验数据行单因素方差分析和SNK法组间比较。结果：猪BMSCs在含有EMPs的培养液中生长良好。对照组以及不同浓度EMPs实验组对细胞黏附的影响无统计学差异。在1h、3．5h、6．5h．各组细胞的伸展率无显著不同。EMPs对BMSCs的促增殖作用呈浓度和时间依赖性．200μg／ml浓度的EMPs从实验的第3天开始．显著促进猪BMSCs的增殖。结论：EMPs对体外培养的猪BMSCs的黏附和伸展无显著影响．200μg／ml浓度的EMPs可显著促进猪BMSCs增殖,为联合应用EMPs和BMSCs修复牙周组织缺损提供了理论依据。     

釉基质蛋白对人骨髓基质细胞生长和黏附的影响

目的探讨釉基质蛋白(enamelmatrixprotein,EMPs)对人骨髓基质细胞黏附、伸展和增殖的影响。方法用全骨髓培养法获得人骨髓基质细胞,各实验组中EMPs的浓度分别为50、100、200、300μg/ml,以不加EMPs作为空白对照组。细胞计数法测定细胞的黏附能力;通过计数高倍视野中伸展的细胞数,计算骨髓基质细胞在1、3、5和7h的伸展率;MTT法检测各组细胞的增殖活性。采用SAS6.12软件对所得数据进行单因素方差分析。结果体外培养的人骨髓基质细胞生长良好;各实验组间及与对照组比较,对细胞的黏附性的影响无统计学差异;各组细胞的伸展率亦无显著性差异;EMPs对人骨髓基质细胞有较明显的促增殖作用,其中200μg/ml浓度组EMPs对骨髓基质细胞的促增殖作用显著(P<0.05)。结论EMPs对体外培养的人骨髓基质细胞的黏附性和伸展率无影响,但可明显促进其增殖。     

壳聚糖温敏凝胶负载釉基质蛋白对骨髓基质细胞的作用

目的:探讨在壳聚糖温敏凝胶支架材料中釉基质蛋白(enamel matrix proteins,EMPs)对骨髓基质细胞(bone marrow stromal cells,BMSCs)增殖和碱性磷酸酶(alkaline phosphate,ALP)活性的影响.方法:合成壳聚糖温敏凝胶并加入适当浓度的EMPs,通过考马斯亮蓝试剂盒检测其对EMPs的缓释作用.用全骨髓培养法获得大鼠BMSCs.含10%胎牛血清的DMEM培养液进行原代培养.含EMPs浓度分别为0、50、100、150μg/mL DMEM培养液培养第3代大鼠BMSCs,MTT法测定各组细胞的增殖活性.MTT法及ALP试剂盒检测大鼠BMSCs在负载100μg/mL EMPs及不负载EMPs的壳聚糖温敏凝胶支架材料中的增殖情况及ALP的活性.采用SPSS11.0软件包对实验数据进行单因素方差分析及两样本t检验.结果:EMPs可在壳聚糖温敏凝胶中持续释放3周以上.DMEM培养液中,50μg/mL EMPs组从实验的第3天开始,显著促进大鼠BMSCs的增殖(P＜0.01).壳聚糖温敏凝胶负载100μg/mL EMPs后.于实验的第3天和第5天明显促进大鼠BMSCs的增殖(P＜0.05)并且在实验的第7天(P＜0.05)和第9天(P＜0.01)显著促进大鼠BMSCsALP水平的提高.结论:壳聚糖温敏凝胶对EMPs具有缓释性,负载EMPs后,可以促进大鼠BMSCs增殖,提高ALP的活性.     

釉基质蛋白对人骨髓基质细胞增殖和矿化的影响

目的:观察釉基质蛋白(enamelmatrix proteins,EMPs)对人骨髓基质细胞(bone marrow stromal cells,BMSCs)细胞周期和碱性磷酸酶(ALP)活性的影响。方法:体外培养人骨髓基质细胞,以200μg/ml EMPs作用于细胞为实验组,以不加EMPs为对照组。流式细胞仪检测EMPs对BMSCs细胞周期的影响,碱性磷酸酶试剂盒检测ALP的活性。结果:体外培养的人BMSCs生长良好,200μg/ml浓度的EMPs能提高细胞的ALP活性;影响细胞周期变化,EMPs组S期细胞数目较多,为19.48%,而对照组S期细胞数目为9.89%。结论:釉基质蛋白可促进人骨髓基质细胞的分化,影响细胞周期变化。     

重组猪釉原蛋白对人牙龈上皮细胞生物学活性的影响

目的:探讨重组25kDa猪釉原蛋白(recombinant porcine amelogenin,rPAm)对人牙龈上皮细胞(human gingival epithelial cells,HGEC)黏附、增殖和迁移的影响。方法:采用不同浓度的rPAm(0、5、10、20μg/mL)刺激第2代HGEC,在相应的时间点,应用细胞计数法测定黏附和增殖的效果,以创面愈合模型测定细胞迁移效果,采用GraphPad Prism软件对数据进行统计学分析。结果:在黏附实验中,rPAm对HGEC的黏附有抑制作用,且效果随着时间的递增和rPAm浓度的增高而加强;在增殖实验中,随着时间的递增和rPAm浓度的增高,HGEC增殖能力下降;在迁移实验中,rPAm能抑制HGEC迁移,20μg/mL效果最好,24h后呈现剂量和时间依赖性。结论:rPAm能显著抑制人牙龈上皮细胞的黏附、增殖和迁移,存在剂量和时间依赖关系。     

蜂胶对人牙周膜成纤维细胞作用的实验研究

目的:探讨蜂胶水提液对人牙周膜成纤维细胞(HPDLFs)生长增殖、骨向分化的影响,寻找蜂胶促增殖及分化作用的最佳浓度。方法:将不同浓度的蜂胶水提液加入体外培养的HPDLFs中,设置阳性对照和空白对照,通过检测HPDLFs的增殖(MTT法)、碱性磷酸酶活性及骨钙素含量,观察蜂胶水提取液对HPDLFs的增殖和分化的影响。结果:10~150μg/ml浓度的蜂胶水提液对体外培养HPDLFs的增殖均有促进作用,100μg/ml浓度时促增殖作用最明显。10~200μg/ml蜂胶水提液均可提高HPDLFs的碱性磷酸酶活性、促进HPDLFs的骨钙素合成(P<0.05),其中以100μg/ml蜂胶水提液作用最显著(P<0.01)。结论:低浓度的蜂胶水提液能促进HPDLFs增殖和向成骨细胞方向分化。     

表没食子儿茶素没食子酸酯对人牙周韧带细胞增殖作用的研究

目的 探讨表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)对人牙周韧带细胞(human periodontal liga-ment cells,hPDLC)体外培养是否存在增殖作用。方法 在体外培养的hPDLC中分别加入50μg/ml、100μg/ml、200μg/ml、400μg/ml和800μg/ml不同浓度的EGCG作为实验组,对照组在普通培养基中培养。两组均在孵育24 h、48 h、72 h后通过MTT法测定细胞增殖情况。结果 在各时间段中,50μg/ml浓度组细胞的吸光度与对照组相比无明显差异(P>0.05),100μg/ml、200μg/ml、400μg/ml和800μg/ml 4个浓度EGCG组细胞的吸光度较高,显著高于对照组(P<0.05)。说明EGCG对人牙周韧带细胞的最小显效浓度是100μg/ml,在100～800μg/ml浓度范围内可明显促进hPDLC的增殖活动。结论 EGCG对hPDLC的增殖具有明显的促进作用。     

釉基质蛋白影响人牙囊细胞生物学特性的实验研究

目的:观察釉基质蛋白(EMPs)对牙囊细胞(HDFC)增殖及碱性磷酸酶(ALP)活性的影响。方法:利用酶消化联合组织块培养法获得HDFC。以一定浓度的EMPs作用于牙囊细胞,通过四唑盐比色法和酶动力学方法检测对细胞增殖及ALP活性的变化。结果:EMPs对HDFC具有促增殖作用,其中100mg/L的EMPs的促增殖作用最显著,其促增殖作用可持续至第7天;100mg/L的EMPs可上调ALP活性。结论:EMPs可促进HDFC增殖,影响其ALP活性,可能在牙囊细胞的诱导分化中起重要作用。     

牙龈卟啉单胞菌牙龈蛋白酶K对人牙周膜细胞增殖的影响

目的 探讨牙龈卟啉单胞菌牙龈蛋白酶K对人牙周膜细胞增殖的影响.方法 组织块法体外培养人牙周膜细胞,不同浓度的牙龈蛋白酶K作用于人牙周膜细胞24h,对照组为无牙龈蛋白酶K作用的人牙周膜细胞.浓度为12.5μg/ml牙龈蛋白酶K分别作用于人牙周膜细胞24、48、72、96h,对照组也培养相同的时间.采用MTT法检测人牙周膜...     

Effect of Emdogain on human periodontal fibroblasts in an in vitro wound-healing model

OBJECTIVE: The aim of this study was to evaluate the influence of Emdogain (EMD) on cultured gingival fibroblasts, periodontal ligament fibroblasts and dermal fibroblasts, using an in vitro model of wound healing. BACKGROUND: Enamel matrix derivative has been demonstrated to promote periodontal regeneration. However, the precise mechanisms by which this agent acts are still unclear. METHODS: The effect of EMD on proliferation of the cells was studied using subconfluent cultures of gingival fibroblasts and periodontal ligament fibroblasts. The cells were made quiescent overnight and then stimulated with various concentrations of EMD (10, 50, 100 and 150 microg/ml) for 24 h. Negative and positive controls were cells cultured in media containing 0.2% and 10% fetal calf serum (FCS). The DNA synthesis was measured by the cellular uptake of [3H]thymidine. For in vitro wounding the cells were cultured, wounded and stimulated with 0.2% FCS, 10% FCS and EMD at a concentration of 20 microg/ml. The percentage of wound fill after treatment was measured after d 1, 4, 6, 12 and 16. The proliferation of cells was also calculated by the extent of incorporation of crystal violet. RESULTS: The results demonstrated that cells in vitro fill an empty space by a combination of proliferation and cell migration. The most rapid closure of a wound area occurred where both proliferation and migration can occur as was seen when wounded cultures were maintained in 10% FCS or at a concentration of 20 microg/ml EMD which promoted proliferation. CONCLUSIONS: Therefore, EMD appears to exert an influence on cells that is compatible with improved wound healing.     

Effect of nicotine-treated epithelial cells on the proliferation and collagen production of gingival fibroblasts

BACKGROUND, AIMS: Several in vitro and in vivo studies have indicated that tobacco smoking may be an important risk factor for the development and severity of inflammatory periodontal disease. METHOD: In the present study, we developed an in vitro model to study the interactions between nicotine-treated epithelial cells (EC) and gingival fibroblasts (GF) derived from the same patient. EC were treated with nicotine concentrations varying from 1 microg/ml to 500 microg/ml and their effect on different functions of GF was studied. The proliferation of GF was evaluated by the incorporation of 3H-thymidine. A dose-dependent inhibition was observed with nicotine concentrations > or =100 microg/ml. Similar results were observed when studying the total protein synthesis of GF by incorporation of 3H-proline into non-dialyzable material. RESULTS: When collagen production was evaluated by 3H-proline incorporation into collagenase-sensitive protein, a dose-dependent reduction was observed: the degree of inhibition varied from 25% with 50 microg/ml nicotine, to almost 60% with 500 microg/ml. Interestingly, the production of non-collagenous proteins decreased by almost 50% only when EC were treated with the highest concentration of nicotine. CONCLUSIONS: The results suggest that epithelial cells, acting as mechanical barrier, can reduce but not completely eliminate the deleterious effect of nicotine on gingival fibroblasts.     

Inhibition of the proliferation of human periodontal ligament fibroblasts by hyaluronidase

Hyaluronan (HA) exists in various living tissues as one of the major matrix macromolecules, and is well known to play an integral role in cell differentiation and proliferation. The present study was conducted to elucidate whether or not the proliferation of periodontal ligament (PDL) cells are affected specifically by the degradation of HA by hyaluronidasze (HAase). Human PDL fibroblasts were isolated and cultured with and without 15-150U/ml bovine testicular HAase from 1 to 11 days after seeding. The cells were also cultured with anti-CD44 antibody of 2 microg/ml. For the control against the anti-CD44 antibody treatment, 2 microg/ml IgG was used. The HA-dependent pericellular matrix was visualized by particle-exclusion assay. The number of cells was counted by MTT assay during the proliferation. The mRNA levels of HA synthases (HASs), HAases (HYALs) and CD44s were examined by a quantitative real-time PCR analysis. The cell proliferation was inhibited by the treatment with HAase and anti-CD44 antibody in cultured PDL fibroblasts. HASs mRNAs were down-regulated, whereas HYALs mRNAs were up-regulated significantly by the treatment with HAase and anti-CD44 antibody. The CD44s mRNA level exhibited no significant changes. These results suggest that HA may contribute to modulate the proliferation of cultured human PDL cells through a CD44-mediated mechanism.     

四环素和多西环素对人牙周韧带细胞生物学活性的影响

目的探讨四环素及多西环素对体外培养的人牙周韧带成纤维细胞(HPDLCs)的生物学作用。方法将不同浓度的2种药物(1、5、20、100、500、2500滋g/ml)加入体外培养的HPDLCs中,共孵育2d后,在倒置显微镜下观察其对细胞形态的影响,并用MTT法、考马司亮蓝法及3H-TdR掺入法,分别检测其对细胞的增殖活性、蛋白合成及DNA合成的影响。结果在1～100滋g/ml的浓度范围内,2种药物对细胞形态无影响。在20～100滋g/ml的浓度范围内,四环素显著促进HPDLCs的增殖及生物合成(P﹤0.01),而多西环素只在20滋g/ml时,能显著促进细胞DNA的合成(P﹤0.01)。当2种药物的浓度增至2500滋g/ml时,不仅使细胞的镜下形态发生明显改变,而且严重抑制细胞的生物学活性。结论在合适的浓度范围内,四环素能显著促进HPDLCs的增殖及生物合成,多西环素的促进作用不明显,而浓度过高时两者均具有细胞毒性。     

Phenytoin and cyclosporin A suppress the expression of MMP-1, TIMP-1, and cathepsin L, but not cathepsin B in cultured gingival fibroblasts

BACKGROUND: Fibroblasts are known not only to synthesize and secrete extracellular matrix proteins, but also to degrade them for connective tissue remodeling. Drug-induced gingival overgrowth is characterized by a massive accumulation of extracellular matrix components in gingival connective tissues. Although some previous reports suggested that causative drugs stimulated the fibroblast proliferation, the results are not conclusive yet. In this study, we hypothesized that drug-induced gingival overgrowth could be a consequence of impaired ability of matrix degradation rather than an enhanced proliferation of gingival fibroblasts induced by these drugs. METHODS: Normal human gingival fibroblasts were cultured with or without either 20 microg/ml of phenytoin or 200 ng/ml of cyclosporin A. Total RNA and cellular proteins were collected every day for RT-PCR analyses and for measuring lysosomal enzyme activity. In addition, an immunohistochemical study was performed to detect lysosomal enzymes in cells from enlarged gingiva of the patients with phenytoin-induced gingival overgrowth. RESULTS: RT-PCR analyses revealed that these drugs suppressed the expression of MMP-1, TIMP-1, and cathepsin L, but not that of cathepsin B in a time-dependent manner. Then, we measured the activity of lysosomal enzymes and cathepsin B and L. The results indicated that although cathepsin B activity was not observed to be impaired, regardless of the drugs used in these cells, both total and active forms of combined activity of cathepsins B and L were suppressed in a time-dependent manner. CONCLUSIONS: The results indicate that, besides suggested effects of these drugs on gingival fibroblasts and/or on accumulated cells in the gingival tissues, extracellular matrix-degrading ability, particularly that by cathepsin L, is also suppressed by cyclosporin A and phenytoin in gingival fibroblasts, and that lysosomal enzyme plays an important role in the pathogenesis of drug-induced gingival hyperplasia.     

Outer membrane vesicles from Porphyromonas gingivalis affect the growth and function of cultured human gingival fibroblasts and umbilical vein endothelial cells

BACKGROUND: The purpose of this study was to examine the effects of outer membrane vesicles (OMV) obtained from Porphyromonas gingivalis (Pg) on the growth and function of human gingival fibroblasts (HGF) and human umbilical vein endothelial cells (HUVEC). METHODS: OMV were obtained from a cell-free growth medium of Pg ATCC 33277 by 40% NH2SO4 precipitation and ultracentrifugation. Cell proliferation was measured by 3H-thymidine incorporation into growing HGF and HUVEC. Endothelial cell function was determined by their capacity to form a network of capillary tubes on an extracellular matrix (ECM). RESULTS: Proliferating HGF and HUVEC demonstrated a significant dose-dependent inhibition of 3H-thymidine uptake when cultured with 0 to 40 microg/ml of OMV protein. HGF and HUVEC showed an IC50 of growth of about 9.0 microg/ml and 4.5 microg/ml of OMV protein, respectively. Capillary tube formation by HUVEC cultured on an ECM was suppressed by 70% to 80% with 5 microg/ml OMV protein after 18 hours of incubation. The presence of proteolytic enzymes in the OMV did not contribute to capillary tube disruption, since blocking enzyme activity with specific inhibitors did not reduce the suppression of capillary tube formation. After heating at 90 degrees C for 5 minutes, OMV significantly lost their capacity to suppress capillary tube formation. CONCLUSIONS: OMV significantly inhibit the proliferation of cultured HGF and HUVEC in a dose-dependent manner. OMV suppressed the capillary tube formation by cultured HUVEC. The factor(s) appeared to be a protein and not endotoxin because its inhibitory activity was markedly reduced by heat inactivation. These studies suggest that OMV contribute to chronic periodontitis by suppressing cell proliferation and revascularization in periodontal tissues.