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.     

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     

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.     

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.     

人牙龈成纤维细胞对甲硝唑的跨膜转运

目的：研究人牙龈成纤维细胞对甲硝唑的跨膜转运，并初步探讨转运的影响因素及牙周局部给药和人工种植牙给药的可行性。方法：将人牙龈成纤维细胞与甲硝唑共同孵育1、5、10、15min后，弃去细胞外药液，收集各时间点细胞，用高效液相色谱法测定细胞内药物含量，用考马斯亮蓝法测定细胞蛋白总量。结果：（1）人牙龈成纤维细胞内甲硝唑含量分别为0．940±0．408ng／ug（药物浓度：20ug／ml）和1．977±0．266ng／ug（药物浓度：40ug／ml）。不同加药浓度和孵育时间下，甲硝唑的转运量差异有显著性（P〈0．01）。（2）高效液相色谱法可以准确测定细胞内甲硝唑的含量。结论：（1）人牙龈成纤维细胞具有转运甲硝唑的能力，药物浓度和药物孵育时间会影响转运量，这证实了牙周局部给药和人工种植牙给药的可行性。（2）高效液相色谱法可以准确，灵敏地测定细胞内药物的含量。     

自酸蚀牙本质粘接剂对人牙髓成纤维细胞的毒性作用

目的比较和评价自酸蚀粘接剂XenoⅢ（XO）和Adper Prompt（AP）以及全酸蚀粘接剂Single bond2（SB）三者的细胞毒性大小。方法将3种牙本质粘接剂XO、AP和SB涂布于直径为5.0 mm、厚度为0.5 mm的牙本质圆片的两面,置于DMEM培养液中获得材料的浸提液,然后将培养液稀释成100.0%、50.0%、25.0%和12.5%四种体积分数。选用组织块法体外原代培养人牙髓成纤维细胞,并将不同体积分数的材料浸提液与第5代人牙髓成纤维细胞共同培养,通过MTT法评价材料24、72、120 h的细胞毒性。结果牙本质粘接系统XO、AP和SB在体外对人牙髓成纤维细胞均有一定程度的细胞毒性,两种自酸蚀粘接剂XO和AP的细胞毒性明显低于全酸蚀粘接剂SB,其差异有统计学意义（P<0.05）。结论自酸蚀牙本质粘接剂XO和AP的细胞毒性小于全酸蚀牙本质粘接剂SB。     

Effect of inflammation upon human gingival oxidative metabolism

Cytochrome oxidase and NADH cytochrome c reductase activities were analyzed biochemically in gingival biopsy specimens obtained from 22 male patients (age 23–72) undergoing periodontal treatment. Histologically, 13 specimens exhibited mild inflammation, while 9 showed more severe inflammatory responses. Cytochrome oxidase activity was significantly greater in the mildly inflamed than in markedly inflamed tissue samples. NADH cytochrome c reductase activity on the other hand was not significantly altered by the increasing degree of inflammation. The possible implication of the effect of inflammation upon oxidative enzymes is discussed in relationto degenerative and proliferative changes occurring in both types of tissue.     

Chlorhexidine-induced changes to human gingival fibroblast collagen and non-collagen protein production

BACKGROUND: Chlorhexidine (CHX) has been used extensively as an adjunctive therapy in the treatment of periodontal disease. It is well known that chlorhexidine is toxic to bacteria, but recent evidence has suggested that chlorhexidine may also have deleterious effects on gingival fibroblast proliferation as well as collagen and non-collagen protein production in cell culture. The purpose of this study was to examine the effects of chlorhexidine on gingival fibroblast proliferation as well as collagen and non-collagen protein production in cell culture. METHODS: Human gingival fibroblasts were incubated in MEM containing chlorhexidine concentrations ranging from 1 microM to 1300 microM at 37 degrees C for 1, 5, or 15 minutes. Control cells received an equal volume of MEM without chlorhexidine for similar times at 37 degrees C. Following incubation, the media were removed, cells washed twice with MEM supplemented with 10% FBS, and fibroblasts in treatment and control groups were allowed to recover in the same media for 24 hours. RESULTS: In all strains, cellular proliferation was dependent on the concentration of solubilized chlorhexidine in cell culture but independent of the duration of chlorhexidine exposure. The average inhibitory concentration necessary to reduce cellular proliferation by 50% (ID50) was 222.1 microM. In regard to collagen and non-collagen protein production, fibroblasts exposed to chlorhexidine concentrations (1 microM) well below the ID50 had a 65% reduction in collagen production and a 57% reduction in noncollagen protein production. CONCLUSIONS: These results suggest that chlorhexidine will induce a dose dependent reduction in cellular proliferation and that concentrations of chlorhexidine that have little effect on cellular proliferation can significantly reduce both collagen and noncollagen protein production of human gingival fibroblasts in vitro. Hence, the introduction of commercially available concentrations (0.12%) or diluted commercial concentrations (as low as 0.00009%) of chlorhexidine to surgical sites for short periods of time prior to wound closure can conceivably have serious toxic effects on gingival fibroblasts and may negatively affect wound healing.     

Modulation of human gingival fibroblast cell metabolism by methyl mercaptan

Methyl mercaptan (CH3SH) is a malodorous compound whose levels are elevated in mouth and crevicular air of individuals with active periodontal disease. Since it may play a role in the disease process, its effects were evaluated using human gingival fibroblast cultures and viable porcine unkeratinized oral mucosal tissue sections. Results showed that the protein content of CH3SH-exposed cell cultures pulsed with [14C]-labelled glycine and proline was decreased by approximately 25%. Furthermore, this deleterious effect was irreversible in test cultures subsequently incubated for 24 h in a control 95% air/5% CO2 mercaptan-free environment. The supporting slab-gel electrophoresis profiles yielded evidence that exposure to CH3SH caused an alteration in collagen metabolism and a pooling of Type I procollagens. In addition, DNA synthesis was suppressed in CH3SH-exposed cultures by 44.1% at the 24 to 26 h peak of DNA synthesis. This is a true inhibition and not a shift in peak of maximum DNA synthesis as the shape and location of time-course curves of control and test systems is very similar. Proline transport study using [14C]-proline indicated a reduction in proline transport in the range of 40 to 50% in cultures exposed for 24 to 30 h to CH3SH. Significantly even 15 min exposure to 6.7 ng CH3SH/ml of incubating atmosphere suppressed proline transport by approximately 24%. This indicates that even brief exposure to low concentrations of CH3SH has a significant adverse effect on proline transport. Fluorescent staining of tissue sections exposed to mercaptan indicated that the agent elevated the number of cells stained with vital dye.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytotoxicity of halothane on human gingival fibroblast cultures in vitro

Recently halothane has been reported to be the most suitable alternative to chloroform in dissolving gutta-percha. Periapical tissue toxicity of halothane is not completely known. In this study gutta-percha dissolved by halothane was evaluated with the almar blue dye assay using human gingival fibroblast cultures. The cytotoxic effects of halothane on human gingival fibroblasts depended on the exposure dose, frequency, and duration. A reduced concentration and smaller amount of gutta-percha solvents may minimize the cytotoxic effects on host tissues.     

Enamel matrix derivative stimulates human gingival fibroblast proliferation via ERK

Emdogain, a formulation of Enamel Matrix Proteins, is used clinically for periodontal regeneration to stimulate PDL (periodontal ligament), cementum, and bone formation. Its effects on gingival fibroblasts and tissue have not been thoroughly studied. Therefore, we investigated the mechanisms by which Emdogain affects the cell cycle of human gingival fibroblasts. Without serum, Emdogain (50 microg/mL) induced human gingival fibroblast entry into the S phase and DNA synthesis, but not completion of the cell cycle. With low serum concentrations (0.2-0.5%), Emdogain synergistically induced completion of the cell cycle, resulting in increased cell numbers. The mitogenic response to Emdogain depended on Extracellular Regulated Kinase (ERK) activation, which occurred in two waves, peaking after 15 min and 4 to 6 hrs, since it was abolished by U0126, a specific MAPK inhibitor. Inhibition of the second wave was sufficient to abrogate mitogenesis. This study characterized the mitogenic effect of Emdogain on primary human gingival fibroblasts, its cooperation with serum growth factors, and the key mediatory role of the ERK cascade.     

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.     

Effect of inflammation upon human gingival cyclic AMP metabolism

The effect of the increasing degree of human gingival inflammation on adenylate cyclase (basal, fluoride stimulated) and low Km and high Km cAMP phosphodiesterase activities were evaluated in separate studies. Human gingival biopsies were classified by the Löe Bleeding Index as (1) mildly, (2) moderately, and (3) markedly inflamed. Basal and F stimulated adenylate cyclase (cAMP synthesis) activities were found to be unaltered by the increasing degree of inflammation when the data were expressed on either a mg wet wt, or mg protein basis. A significant loss of F stimulated adenylate cyclase (mg protein) activity was observed in the moderately inflamed group when the data were compared with either the mildly or markedly inflamed groups of tissue. The low Km, and high Km cAMP phosphodiesterase activities (cAMP degradation) were found to he unaffected by gingival inflammation. This suggests that neither cAMP synthesis, nor degradation are stimulated in human gingiva by inflammation.     

Effect of autologous and allogenic platelet-rich plasma on human gingival fibroblast function

Oral Diseases (2012) 18 , 494–500 Objective: Platelet‐rich plasma (PRP) has been proposed as a method of delivering growth factors to enhance regeneration. The aim of this study was to investigate the use of autogenous and allogenic PRP and platelet‐poor plasma (PPP) on migration and proliferation of human gingival fibroblasts in vitro. Methods: Various concentrations of PRP, as well as PPP, were prepared from autologous and allogenic sources and applied to primary gingival fibroblasts. Migration was determined by assessing the fibroblast response to a concentration gradient. ³H‐thymidine incorporation and crystal violet colorimetric assays were utilized to assess DNA synthesis and proliferation. Results: Platelet‐rich plasma provides a significant migratory stimulus to gingival fibroblasts. Furthermore, the various concentrations of PRP (50%, 20% and 10%) do not promote DNA synthesis in the short term (24 h), but over the longer term (5 days) they stimulate an increase in cell proliferation. Compared with PPP, PRP was superior in terms of encouraging migration, but was inferior in terms of promoting DNA synthesis and cell proliferation. No difference was noted between the autologous and allogenic PRP preparations on cell function. Conclusion: Both PPP and PRP promote gingival fibroblast migration and proliferation in vitro, without differences between preparations obtained from autologous and allogenic sources.     

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。