人牙龈成纤维细胞体外降解胶原能力的测定

目的 :观察体外培养的健康人牙龈成纤维细胞对鼠Ⅰ型胶原降解的影响和时间效应。方法 :在 6孔板底制备胶原膜 ,取固定数量的对数生长期健康人牙龈成纤维细胞接种于胶原膜中央 (2 5× 10 3/孔 ) ,分别于孵育 2 4、48、72h后消化弃去细胞 ,考马斯亮蓝液染色胶原膜 ,显微镜下测定降解区和非降解区的吸光度来判定不同时间段的胶原降解量。结果 :3例标本来源细胞的结果基本一致 ,表现为随时间的延长 ,胶原降解量也相应增加 ,2 4、48、72h的胶原降解量分别约为 2 0 %、40 %、60 %。结论 :健康人牙龈成纤维细胞对鼠Ⅰ型胶原有降解作用 ,而且有一定时间效应 ,这可能是细胞本身的功能变化所为 ,而非细胞数量变化的结果     

Estrogen and extracellular matrix influence human gingival fibroblast proliferation and protein production

BACKGROUND: A paucity of information exists concerning how estrogen affects cellular function in the gingiva of women. In this study, the behavior of human gingival fibroblasts was examined in the presence of a potent estrogen, estradiol. METHODS: Quiescent, premenopausal gingival fibroblasts were incubated in the presence and absence of estradiol (1 nM) and/or raloxifene (100 nM). Cell number was determined and cell cycle analyzed using a flow cytometer. Collagen and non-collagen production in cell cultures grown on various extracellular matrices were determined using a radioactive microassay which measures collagenase-digestible and collagenase-resistant radiolabeled proteins. To ascertain if the gingiva contained specific estrogen-sensitive cell populations, a fluorescence-activated cell sorter was used to detect, sort, and enrich fibroblast populations responsive to estrogen. RESULTS: Cellular proliferation and the number of cells entering the S-phase of the cell cycle were significantly increased in mass cultures of fibroblasts stimulated by estradiol. Raloxifene did not antagonize the action of estradiol on cell proliferation. In regard to protein production, estradiol significantly reduced collagen production on plastic and collagen IV matrices; whereas non-collagen protein production on plastic and collagen I matrices was significantly reduced. Cell sorting of mass fibroblast populations revealed that, on average, 45% of the cells from the resident population selectively accumulated the estrogen probe. These sorted and estrogen-sensitive enriched cell populations proliferated in the presence of 1 nM estradiol, whereas the sorted, estrogen-deficient enriched fibroblast populations did not proliferate when incubated with 1 nM estradiol. CONCLUSIONS: These data indicate that estradiol can induce cellular proliferation while depressing protein production in cultures of human, premenopausal gingival fibroblasts. This cellular proliferation appears to be the result of a specific population of cells within the parent culture that responds to physiologic concentrations of estradiol.     

Acoustic energy affects human gingival fibroblast proliferation but leaves protein production unchanged

BACKGROUND, AIMS: Sonic toothbrushes are well-established in oral home care for plaque removal; however, the effects of low frequency acoustic (sonic) energy released from sonic toothbrushes to the cells of the periodontium have not been investigated. The purpose of this study was to evaluate the effects of sonic energy on human gingival fibroblast proliferation and protein production in cell culture. METHODS: Direct and indirect transfer calibration studies found the fundamental frequency of the Sonicare sonic toothbrush to be 261 hertz (Hz) with amplitudes ranging from 70 to 104 decibels (dB) in the human periodontium. Using an in vitro delivery system, which coupled a signal-wave generator with a bone transducer to mimic the energy delivered by the Sonicare toothbrush, the effects of signal, amplitude and duration were evaluated longitudinally using a gingival fibroblast cell culture model. 8 strains of fibroblasts isolated from healthy human gingiva were seeded at 30,000 cells/35 mm culture dish in minimum essential medium supplemented with 10% fetal bovine serum. To ascertain the relationship of the amplitude and the duration of sonic stimulation to cellular proliferation, gingival fibroblasts were subjected 2x daily to 261 Hz sound at various amplitudes (67-97 dB) for 0, 15, 30, 60, and 120 s on days 1, 3, 5, 7, and 10. RESULTS: It was found that either 30 or 120 s of sound exposure for 10 days of treatment had significant effects on cell proliferation in comparison to control cultures. Specifically, at day 10, 87 dB at 261 Hz for 30 s 2x daily resulted in a 25.5% increase in cell number (p<0.001), whereas 87 dB at 261 Hz for 120 s twice daily caused a 30.9% decrease in cell number (p<0.001) when compared to control cultures. When cells are stimulated under optimum acoustic conditions for 10 days, there was no difference between the treatment and control groups for collagen (p=0.897) or noncollagen (p=0.697) protein production. CONCLUSIONS: Sonic energy has been shown to both increase and decrease cellular proliferation depending on exposure time; however, during optimum sound-induced conditions for cellular proliferation, sonic energy had no effect on fibroblast protein production. These data suggest that sonic energy can affect the behavior of cells in culture. Further research into the mechanisms of these changes will provide important information for manipulating cellular behavior.     

Mechanism of endotoxin inhibition of human gingival fibroblast attachment to type I collagen

Bacterial endotoxin inhibits the attachment of human gingival fibroblasts to collagen. The present study attempted to elucidate the possible mechanism of this inhibition. Two mechanisms were considered: direct toxicity to the cells and steric interference. Collagen substrates were prepared by rat type I collagen being air-dried in the wells of 24 multi-well plates. Experimental collagen substrates were treated with 50 micrograms of endotoxin/well, while untreated collagen substrates served as controls. Two mL of cell suspension (10(4) cells/mL) was added to each well, and these were incubated at 37 degrees C for two h. The average cell number/mm2 attached to experimental and control substrates was determined. Cell attachment to endotoxin-treated collagen was inhibited by 78%, compared with that to untreated collagen. The washing of the endotoxin-treated collagen for two h did not affect the inhibition of cell attachment, whereas after 24 h of washing, cell attachment was inhibited by 54%, compared with that to untreated collagen. Pre-incubation of the cells in endotoxin for two h did not affect their attachment to collagen. The addition of fetal calf serum (15%) to the experimental system completely reversed the inhibition of fibroblast attachment to endotoxin-treated collagen. These findings suggest that endotoxin interferes with fibroblast attachment to collagen through a steric phenomenon, possibly by blocking the binding sites on the collagen molecule recognized by the membrane receptor for collagen.     

不同来源人牙龈成纤维细胞体外降解胶原能力研究

目的 :体外培养条件下比较健康人及侵袭性牙周炎患者的牙龈成纤维细胞对鼠I型胶原降解的影响和差异。方法 :在 6孔板底制备胶原膜 ,取固定数量的对数生长期健康人及侵袭性牙周炎患者的牙龈成纤维细胞接种于胶原膜中央 ,分别于孵育 2 4、48、72h后消化弃去细胞 ,考马斯亮兰液染色胶原膜 ,显微镜下测定降解区和非降解区的光密度来比较不同时间段各组细胞的胶原降解量。结果 :健康人和侵袭性牙周炎各 3例标本来源细胞的结果基本一致 ,表现为随时间的延长 ,胶原降解量相应增加 ;而各时间段病变组的胶原降解量较健康组明显增加(P <0 .0 5 )。结论 :健康人和侵袭性牙周炎的牙龈成纤维细胞对鼠I型胶原均有降解作用并呈现出时间效应 ,而且病变组的作用更强。     

Human gingival fibroblast production of interferon

Three human gingival fibroblast cell lines were used to determine whether they could be induced by a synthetic RNA and superinduced by metabolic inhibitors to produce interferon (IFN-beta). When established procedures were followed for human fetal or newborn skin fibroblast cell lines, the adult gingival fibroblasts produced comparable amounts of IFN-beta. It was shown that the superinducers alone would not cause an IFN-beta production response, and that the absence of serum in the production medium also inhibited the production of IFN-beta. The effect of IFN-beta on cell growth was carried out in T-flasks seeded with 10(5) HEp-2 cells. After one and two wk, the cells of triplicate control flasks and triplicate flasks containing various dilutions of the production media were enumerated to determine a cell multiplication inhibition (CMI) value. A correlation between the IFN-beta content and the CMI effect, however, was not obtained, and it was concluded that other CMI agents, possibly more potent than the IFN-beta, were being produced by the stimulated human gingival fibroblasts. Cell protein assays which gave a high ng/cell protein content correlated with TEM micrographs which showed clusters of complex lysosomes, primarily in cells cultured in the IFN-beta-containing nutrient. However, since commercial IFN-beta initiated no such lysosomal response, it was further concluded that the complex lysosomes were due to CMI agent(s) other than IFN-beta.     

Nitric oxide inhibits androgen receptor-mediated collagen production in human gingival fibroblasts

Lin S‐J, Lu H‐K, Lee H‐W, Chen Y‐C, Li C‐L, Wang L‐F. Nitric oxide inhibits androgen receptor‐mediated collagen production in human gingival fibroblasts. J Periodont Res 2012; 47: 701–710. © 2012 John Wiley & Sons A/S Background and Objective: In our previous study, we found that flutamide [an androgen receptor (AR) antagonist] inhibited the up‐regulation of collagen induced by interleukin (IL)‐1β and/or nifedipine in gingival fibroblasts. The present study attempted to verify the role of nitric oxide (NO) in the IL‐1β/nifedipine‐AR pathway in gingival overgrowth. Material and Methods: Confluent gingival fibroblasts derived from healthy individuals (n = 4) and those with dihydropyridine‐induced gingival overgrowth (DIGO) (n = 6) were stimulated for 48 h with IL‐1β (10 ng/mL), nifedipine (0.34 μm ) or IL‐1β + nifedipine. Gene and protein expression were analyzed with real‐time RT‐PCR and western blot analyses, respectively. Meanwhile, Sircol dye‐binding and the Griess reagent were, respectively, used to detect the concentrations of total soluble collagen and nitrite in the medium. Results: IL‐1β and nifedipine simultaneously up‐regulated the expression of the AR and type‐I collagen α1 [Colα1(I)] genes and the total collagen concentration in DIGO cells (p < 0.05). IL‐1β strongly increased the expression of inducible nitric oxide synthase (iNOS) mRNA and the nitrite concentration in both healthy and DIGO cells (p < 0.05). However, co‐administration of IL‐1β and nifedipine largely abrogated the expression of iNOS mRNA and the nitrite concentration with the same treatment. Spearman’s correlation coefficients revealed a positive correlation between the AR and total collagen (p < 0.001), but they both showed a negative correlation with iNOS expression and the NO concentration (p < 0.001). The iNOS inhibitor, 1400W, enhanced IL‐1β‐induced AR expression; furthermore, the NO donor, NONOate, diminished the expression of the AR to a similar extent in gingival fibroblasts derived from both healthy patients and DIGO patients (p < 0.05). Conclusion: IL‐1β‐induced NO attenuated AR‐mediated collagen production in human gingival fibroblasts. The iNOS/NO system down‐regulated the axis of AR/Colα1(I) mRNA expression and the production of AR/total collagen proteins by DIGO cells.     

糖基化终产物对牙龈成纤维细胞增殖和Ⅰ型胶原合成的影响

目的 观察糖基化终产物对牙龈成纤维细胞增殖能力和Ⅰ型胶原合成的影响,探讨糖基化终产物对牙周组织修复功能的作用及在伴有糖尿病牙周炎中的致病机制.方法 采用组织块法培养牙龈成纤维细胞,培养基中加入体外合成的不同浓度的糖基化终产物,甲基噻唑基四唑(MTT)法检测在不同时间段下牙龈成纤维细胞增殖水平的变化,酶联免疫吸附测定法和实时定量反转录聚合酶链法(RT-PCR)分别测定牙龈成纤维细胞合成Ⅰ型胶原的量和表达Ⅰ型胶原mRNA的水平.结果 200 mg/L糖基化终产物作用于牙龈成纤维细胞48、74 h的A值分别为0.016±0.023、0.035±0.008,比其他组A值低(P＜0.05),并且细胞形态发生了改变,细胞由均一的梭形变成圆形、椭圆形、细长不规则条状等,胞质浓缩,细胞间隙增大;糖基化终产物作用72 h后,牙龈成纤维细胞上清液中和胞内Ⅰ型胶原的含量减少(P＜0.05),Ⅰ型胶原mRNA表达下调(P＜0.05).结论 糖基化终产物能抑制牙龈成纤维细胞的增殖、降低其合成Ⅰ型胶原蛋白和表达Ⅰ型胶原mRNA,从而可能削弱了牙周组织的愈合能力.     

Cytokine regulation of gingival fibroblast lysyl oxidase, collagen, and elastin

BACKGROUND: Systemic therapy with cyclosporin A, phenytoin, and nifedipine modulates cytokine levels in human gingival tissues. Functional relationships between altered cytokine levels and gingival extracellular matrix production are partially characterized. The present study investigates in cultured human gingival fibroblasts the regulation of lysyl oxidase, alpha-1 type I collagen, and elastin by selected cytokines that are elevated in drug-induced gingival overgrowth tissues. METHODS: Normal human gingival fibroblasts were cultured and then treated with selected cytokines: interleukin (IL)-1beta, IL-6, platelet-derived growth factor (PDGF)-BB, and basic fibroblast growth factor (bFGF or FGF-2). Cells were harvested at intervals, and changes in lysyl oxidase enzyme activity, and in mRNA levels of lysyl oxidase, alpha-1 type I collagen, and elastin were determined. RESULTS: bFGF reproducibly and significantly decreased human gingival fibroblast lysyl oxidase and alpha-1 type I collagen mRNA levels in a dose- and time-dependent manner; 1 nM bFGF reduced lysyl oxidase and collagen mRNA levels to 53% and to less than 10% of control after 48 hours of treatment. Interestingly, bFGF downregulated lysyl oxidase enzyme activity by 10% to 20%. IL-1, IL-6, and PDGF-BB did not significantly regulate lysyl oxidase enzyme activity, or alpha-1 type I collagen, elastin, and lysyl oxidase mRNA levels under the conditions tested. CONCLUSIONS: Previous studies have shown that modulated levels of bFGF occur in gingiva as a result of certain pharmacologic therapies. The present study suggests that modulated levels of bFGF likely influence gingival connective tissue metabolism.     

Stimulation of human periodontal ligament fibroblast collagenase production by a gingival epithelial cell-derived factor

To examine whether cell-to-cell interactions between human gingival epithelial cells (HGE) and periodontal ligament fibroblasts (PLF) or gingival fibroblasts (GF) take place in the periodontium, the effects on collagenase production by PLF and GF were analyzed after adding several concentrations of HGE-con-ditioned medium (HGE-CM) to PLF or GF culture. Collagenase production by both cell populations was stimulated by adding HGE-CM, which stimulated collagenase production by PLF to a greater extent than that by GF. The HGE-derived stimulatory factor had a molecular mass of approximately 20 kDa, and its stimulant effect was inhibited markedly in the presence of an anti-human interleukin-lα (IL-lα) neutralizing antibody, indicating that the factor was identical to, or antigenically cross-reactive with, IL-lα. These results suggest that epithelial apical migration in the periodontium may occur after interstitial resident cells have released tissue-degrading enzymes, such as collagenase, and damaged the extracellular matrix, once a sufficient amount of IL-lα-like factor for stimulating the production of proteolytic enzyme has been released by HGE in periodontal lesions.     

Composite-induced toxicity in human gingival and pulp fibroblast cells

Objective. The most important requirement for a material to be used in medical applications is its biocompatibility. Dental composite materials come into direct contact with oral tissues, especially gingival and pulpal cells. This study was performed to evaluate possible DNA damage in cells of human origin exposed to dental composites in vitro using a cytogenetic assay. Materials and methods. Two composite resins (Vertise Flow, Kalore) were tested on human gingival and pulp fibroblasts using the acridine orange/ethidium bromide viability staining and alkaline comet assay. Cultures were treated with polymerized composites in two different concentrations (20 mg/ml, 40 mg/ml) for 14 days. Chi-square and Kruskall-Wallis non-parametric test were used for the statistical analysis (p < 0.05). Results. Significant cytotoxicity was observed for 40 mg/ml of Vertise Flow in both cultures, while Kalore (40 mg/ml) showed cytotoxic effect only on human pulp fibroblasts. A significant level of DNA damage was detected for both materials and concentrations, in both cell cultures. Conclusion. If the two cell cultures are compared, the pulp cells were more sensitive to the cyto/genotoxic effects of dental composites. Based on the results, one can conclude that the use of tested materials may cause cellular damage in gingival and pulp fibroblasts in vitro.     

Collagenolysis by human gingival fibroblast cell lines.

Human gingival fibroblast cell lines were initiated in flask cultures from four periodontal patients with the diagnoses of periodontitis (two patients), fibromatosis, and Dilantin hyperplasia. The collagenolytic propensity of these fibroblasts cultivated on collagen-coated cover slips and the inhibitory effect of serum were evaluated by direct microscopic observations.     

Capsular polysaccharide from Actinobacillus actinomycetemcomitans inhibits IL-6 and IL-8 production in human gingival fibroblast

We previously reported that a capsular polysaccharide (CP) from Actinobacillus actinomycetemcomitans Y4 induces bone resorption in a mouse organ culture system and osteoclast formation in mouse bone marrow cultures. However, the effects of A. actinomycetemcomitans Y4 CP on human gingival fibroblasts (HGF) are still unclear. The present study was undertaken to test the hypothesis that A. actinomycetemcomitans Y4 CP alters the production of inflammatory cytokines, such as interleukin-6 (IL-6) and IL-8 by HGF. When HGF were cultured with various concentrations of Y4 CP for 24 h, IL-6 and IL-8 production decreased in a concentration-dependent manner. Y4 CP (100 microg/ml) suppressed the release of IL-6 from 9.09 +/- 0.08 ng/ml to 0.34 +/- 0.21 ng/ml (P < 0.01) and IL-8 production decreased from 3.76 +/- 0.03 ng/ml to 0.09 +/- 0.01 ng/ml (P < 0.01). Y4 CP suppressed 70-80% of the release of IL-6 and IL-8 from HGF stimulated with Y4 lipopolysaccharide (LPS), too. Interestingly, anti-A. actinomycetemcomitans Y4 CP completely inhibited the effect of A. actinomycetemcomitans Y4 CP on IL-6 and IL-8 production from HGF. These results indicate that Y4 CP inhibits the release of IL-6 and IL-8 from HGF, suggesting that A. actinomycetemcomitans Y4 modulates the inflammatory response in periodontitis. Remarkably, this inhibitory effect was reversed by specific anti-A. actinomycetemcomitans Y4 CP suggesting an important relationship between the organism and the humoral host response.     

Inflammatory changes in gingival collagen in the alloxan-diabetic rat

Chronic inflammation was induced by repeatedly injecting ferritin-antiferritin (AgAb) complex into gingiva of normal, untreated and insulin-treated alloxan diabetic rats. Blood glucose levels, the urinary excretion of hydroxyproline, and the salt-soluble, acid-soluble and insoluble collagen levels in gingiva were measured in all groups of rats. Additionally, the gingival tissues were examined by light and electron microscopy. Injection of AgAb into the gingiva of normal rats produced typical chronic inflammatory cell infiltrates, fibroblasts containing profiles of intracellular collagen, disruption of extracellular collagen fibrils and a reduction in collagen concentration primarily affecting the insoluble fraction. The inflamed gingiva of the diabetic rats demonstrated similar changes histologically except that the macrophages were more congested with phagocytosed material and the fibroblasts appeared smaller and less well developed. Unlike the normal rats, the collagen content of the diabetic gingiva was not reduced by inflammation. This loss of the inflammatory effect on gingival collagen content during experimental diabetes was explained by three possibilities: (1) that diabetic fibroblasts had a decreased ability to effect collagen remodeling during inflammation, (11) that the inflammatory cells responsible for gingival collagen loss were less active in diabetes, and (111) that diabetic gingival collagen, because it was more highly crosslinked than normal, resisted the degradative enzymes produced in increased amounts during the inflammatory process.     

人牙龈成纤维细胞系的建立及其生物学特性

目的：建立人牙龈成纤维细胞系并观察其生物学特性。方法：用组织块法培养细胞,用形态学、免疫组化染色、染色体分析等方法鉴定细胞,通过活细胞观察、生长曲线测定及ＭＴＴ比色实验研究细胞体外生长特性。结果：２０例原代培养,成活率９０％。培养细胞呈梭形,胞浆波形丝蛋白阳性,能合成Ⅰ型和Ⅲ型胶原及纤维粘连蛋白,具有正常二倍体核型,体外贴壁快,生长迅速,细胞寿命为（２５０．７±１１３．３）ｄ。结论：本实验建立的细胞系符合人牙龈正常二倍体成纤维细胞系。     

Pluronic polyol effects on human gingival fibroblast attachment and growth

BACKGROUND: Enhanced speed of human gingival fibroblast (HGF) spreading and attachment, as affected by ionic bonding interactions, may facilitate cell orientation and subsequent collagen synthesis to promote early wound healing. The purpose of this study was to determine the in vitro effects of pluronic polyols, a family of widely used surfactants currently used as drug carriers for antibiotic, anti-inflammatory, and anti-neoplastic agents, on the attachment and growth of human gingival fibroblasts (HGF) to dentin and plastic surfaces using established tissue culture techniques. METHODS: Plastic culture wells containing Eagle's minimal essential media (EMEM) with 10% fetal calf serum and Pluronic F-68 or F-127 in concentrations from 1.2 x 10(-2) to 1.2 x 10(-10) M were incubated with HGF and run in replicates of ten. Attached cells were quantified by measuring the optical density of methylene blue-stained cells. Additional experiments were conducted using human dentin sections as a substrate and Pluronic F-68 or F-127 at a concentration of 1.2 x 10(-8) M. In these experiments, HGF were stained with acridine orange and quantified per unit area of dentin by fluorescence microscopy. RESULTS: Attachment and growth of HGF to both plastic and dentin were significantly increased over serum controls by very low concentrations of Pluronic F-68 and F-127 by 30 minutes, with attachment reaching a plateau at 2 hours. CONCLUSIONS: Pluronic polyols, a family of widely used surfactants, in very low dosages may be beneficial in early postsurgical wound healing by facilitating early attachment and enhancing the growth rate of human gingival fibroblasts.