人牙龈成纤维细胞和牙周膜成纤维细胞的培养和生物学特征

目的：探讨采用不同方法建立体外培养人牙龈成纤维细胞和人牙周膜成纤维细胞并对其生物学特性作初步探讨。方法：分别采用消化培养法和组织块培养人的牙龈和牙周膜成纤维细胞。通过倒置显微镜活体观察，以及光镜、透射电镜、免疫组化和生长曲线等方法对其生物学特性作初步观察。结果：消化培养法成功率低于组织块培养法。光镜下和透射电镜下观察两种细胞在形态和结构上相似。免疫组化染色证实此两种细胞均来源于中胚层的纤维母细胞。生长曲线表明牙龈成纤维细胞的倍增时间比牙周膜成纤维细胞稍短，而牙周膜成纤维细胞的增殖活性比牙龈成纤维细胞高。结论：组织块培养法适用于此二种细胞的培养。细胞生物学特征方面有许多相似形，提示这两种细胞来于同一个细胞群。     

维拉帕米对正常牙龈成纤维细胞增殖的影响

目的体外评价维拉帕米对正常牙龈成纤维细胞增殖的影响。方法分离、培养正常牙龈成纤维细胞。用0、0．1、1、10、100μmol／L5个浓度的维拉帕米分别作用于第5代正常牙龈成纤维细胞，采用甲基噻唑基四唑法检测细胞的增殖，流式细胞仪评价细胞生长周期。结果100μmol／L维拉帕米作用66h后，检测A值为0．046，与无药物作用组（A值为0．088）相比差异有统计学意义（P〈0．01）。100μmol／L维拉帕米作用18h后，69％的细胞处于G0-G1期，27％处于S期，而无药物作用组分别为41％和49％。两者间差异有统计学意义（P〈0．001）。结论100μmol／L维拉帕米在体外通过阻滞细胞生长周期抑制正常牙龈成纤维细胞的增殖。     

人牙龈成纤维细胞原代培养方法的比较研究

目的：建立和评价人牙龈成纤维细胞原代培养方法,并观察其生物学特性。方法：分别用组织块法、2种改良酶消组织块法培养人牙龈成纤维细胞（HGF）,用形态学、免疫荧光鉴定细胞来源。通过活细胞观察,MTT比色实验研究细胞体外生物特性。比较3种培养方法培养HGF的效果。结果：细胞抗波形丝蛋白染色阳性,抗角蛋白染色阴性,符合人牙龈成纤维细胞的形态学特征和生物学特性。组织块法、改良酶消组织块法（翻瓶法）、改良酶消组织块法（盖玻片法）的细胞培养成功率分别为26.7%、54%、60%。组织块法和2种改良酶消组织块法间的成功率差异有显著性（P〈0.01）,2种改良酶消组织块法间的成功率差异无显著性（P〉0.05）。结论：本实验建立的细胞系为人牙龈成纤维细胞。2种改良酶消化组织块法可显著提高人牙龈成纤维细胞原代培养成功率。     

内毒素对牙龈成纤维细胞mCD14表达的影响

目的:研究mCD14在牙龈成纤维细胞的膜表面分布及内毒素对mCD14在牙龈成纤维细胞膜表面表达的影响。方法:组织块法培养人牙龈成纤维细胞,利用免疫组化和Western blot方法研究mCD14在牙龈成纤维细胞的膜表面分布,同时观察内毒素对mCD14在牙龈成纤维细胞膜表面表达的影响。结果:免疫组织化学染色实验和蛋白印迹实验结果均表明mCD14在牙龈成纤维细胞的膜表面表达阳性,同时LPS可增强膜表面的mCD14的表达。结论:本实验表明正常人牙龈成纤维细胞可表达mCD14,LPS可以上调膜表面mCD14的表达,从而导致牙周损伤。     

牙龈卟啉单胞菌内毒素对成纤维细胞在纯钛表面附着的影响

目的：研究牙龈卟啉单胞菌内毒素对纤维细胞在纯钛表面附着的影响。方法：通过对种植体周围牙龈卟啉单胞菌内毒素脂多糖的提取、鉴定以及对成纤维细胞的作用，同时结合光镜及电子显微镜观察与统计分析。结果：发现成纤维细胞在牙龈卟啉单胞菌内毒素作用下在纯钛表面生长形态改变，趋动附着能力降低。结论：牙龈卟啉单胞菌内毒素具有抑制成纤维细胞在纯钛表面附着的作用。     

人牙龈和牙周韧带成纤维细胞体外矿化能力的比较研究

目的：研究人牙龈和牙周韧带成纤维细胞体外矿化能力的差异。方法：用组织块法培养同一患者的牙龈和牙周韧带成纤维细胞，取第4-8代细胞用矿化培养液长期培养，倒置显微镜下观察矿化情况，von Kos-sa染色显示钙盐沉积，生化法测定各组碱性磷酸酶活性，扫描电镜及X线能谱法分析矿化结节中钙磷比例。结果：倒置显微镜下观察牙周韧带成纤维细胞可呈复层生长，形成肉眼可见的白色结节，von Kossa染色显示结节内有钙盐沉积，碱性磷酸酶活性测定表明随着矿化培养时间的延长牙周韧带成纤维细胞组ALP活性比牙龈成纤维细胞组增高明显，扫描电镜下表明结节处电子反射增强，X射线能谱分析显示钙化结节内的钙磷比例与矿化组织相似；牙龈成纤维细胞及对照组体外不能形成钙化结节。结论：人牙龈和牙周韧带成纤维细胞体外矿化能力不同。     

Ⅰ、Ⅲ型胶原在牙龈成纤维细胞和牙周韧带细胞中表达的比较研究

目的比较人牙龈成纤维细胞与牙周韧带细胞在胶原基质合成方面的差别,并探讨其意义。方法采用组织块法体外培养人牙龈成纤维细胞与牙周韧带细胞,利用免疫细胞化学技术检测Ⅰ、Ⅲ型胶原在两种细胞中的表达,通过图像分析探讨其表达的差异。结果Ⅰ、Ⅲ型胶原在牙周韧带细胞中表达阳性,在牙龈成纤维细胞中染色较弱。统计学分析显示,Ⅰ、Ⅲ型胶原在牙龈成纤维细胞与牙周韧带细胞的免疫细胞化学染色结果具有显著性差异(P<0.01)。结论牙龈成纤维细胞与牙周韧带细胞胶原基质的合成或分泌存在差异,这种差异可作为鉴别两种细胞的标志之一。     

人牙周膜细胞和牙龈成纤维细胞生物学活性的研究

目的:体外原代培养人牙周膜细胞和牙龈成纤维细胞,并对其生物学活性作初步探讨.方法:采用组织块法原代培养牙周膜细胞和牙龈成纤维细胞,绘制生长曲线,测定二者碱性磷酸酶活性;流式细胞术和免疫组化染色法测定Ⅰ、Ⅲ型胶原、骨形成蛋白的表达情况,观察对比两种细胞的生物学特性的异同.结果:牙龈成纤维细胞原代培养成功率及细胞增殖活性明显高于牙周膜细胞.在牙周膜细胞中,Ⅰ、Ⅲ型胶原均为阳性表达,棕黄色颗粒克满整个胞浆内.在牙龈成纤维细胞中Ⅰ型胶原为弱阳性表达,Ⅲ型胶原阳性表达更弱.牙周膜细胞的ALP水平明显高于牙龈成纤维细胞.牙周膜细胞BMP2表达为强阳性,而牙龈成纤维细胞表达弱阳性.结论:牙周膜细胞具有较强的成骨能力,是理想的牙周组织工程的种子细胞.牙龈成纤维细胞易于培养成活,增殖力强,具有牙周膜细胞的一些特点,组织取材方便,也可作为牙周组织工程的种子细胞.     

人牙龈和牙周韧带成纤维细胞的体外培养及生物学特性研究——细胞形态、生长曲线及碱性磷酸酶活性测定

目的 :建立人牙龈和牙周韧带成纤维细胞体外培养模型并对其生物学特性作初步探讨。方法 :采用组织块法常规条件下分别进行牙龈和牙周韧带成纤维细胞的培养 ,通过光镜、透射电镜、生长曲线及碱性磷酸酶测定等手段对其部分生物学特性进行研究。结果 :两种培养的原代及传代细胞在光镜下细胞排列及结构无明显差别 ,传代培养时牙龈成纤维细胞有接触抑制现象 ,而牙周韧带成纤维细胞则可呈复层生长 ,牙周韧带成纤维细胞排列方向性较明显 ,生长曲线表明牙周韧带成纤维细胞增殖活性高于牙龈成纤维细胞 ;碱性磷酸酶活性测定及染色法显示两者有明显的差别。结论 :体外培养的两种细胞在形态及排列上相似 ,但在细胞亚型的组成上存在差异。     

内毒素对牙龈成纤维细胞增殖活性及分泌基质金属蛋白酶的影响

目的观察内毒素对牙龈成纤维细胞增殖活性及分泌基质金属蛋白酶-1,3量的影响,探讨基质金属蛋白酶在牙周炎致病机制中的可能作用。方法通过改良酶消化组织块法进行牙龈成纤维细胞的原代培养,用内毒素对其进行刺激,运用MTT法观察内毒素对牙龈成纤维细胞增殖活性的影响,通过免疫组化法检测内毒素对牙龈成纤维细胞分泌基质金属蛋白酶-1,3的影响。结果MTT结果显示,低浓度内毒素在短期内可以促进牙龈成纤维细胞的增殖,但随着时间的延长,也表现为对细胞的毒性作用,抑制其增殖;高浓度内毒素抑制牙龈成纤维细胞的增殖;免疫组化研究结果表明,未受LPS刺激的牙龈成纤维细胞MMP-1,3表达弱阳性,受刺激后阳性表达增强,在12h时达到顶峰,且MMP-3的整体表达较MMP-1弱。结论LPS可以影响牙龈成纤维细胞的增殖活性;LPS可以促进牙龈成纤维细胞对基质金属蛋白酶的分泌,加快牙周细胞外基质的降解,加速牙周炎的进程。     

Fabrication of cultured oral gingiva by tissue engineering techniques without materials of animal origin

BACKGROUND: Cultured gingival substitute has been found to be a useful graft material for treatment of gingival recession. However, such substitutes include xenograft derivative materials that involve concomitant risk of viral contamination. To eliminate this risk, we designed new gingival substitutes made of recombinant human collagen types I and III sponges and cultured these substitutes in animal-free media (HFDM-1). METHODS: Gingival fibroblasts were seeded onto sponges of type I or III recombinant collagen. These sponges were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), HFDM-1 with 2% human serum (HS), or HFDM-1. Fibroblast proliferation in these samples was compared using the cell-counting kit assay. Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) released into the cultured media were examined by enzyme-linked immunosorbent assay. RESULTS: The fibroblasts proliferated significantly in all six combinations of collagen and medium types. The fibroblast growth rate after 9 days of culture was equal between HFDM-1 with 2% HS and DMEM with 10% FBS. The type III collagen sponge showed a higher fibroblast growth rate than the type I sponge. VEGF concentrations in HFDM-1 with 2% HS were higher than those in other media. The highest HGF levels were detected in DMEM with 10% FBS. CONCLUSIONS: The new cultured gingival substitute containing no animal-derived materials produced good cell proliferation and VEGF release. The results suggested that the substitute may provide a new tool for the treatment of gingival recession.     

Human periodontal ligament and gingival fibroblast response to TGF-β1 stimulation

Abstract. The purpose of this study was to measure the time-sequence response of RNA and protein synthesis to transforming growth factor-β1 (TGF-β1) by human periodontal ligament (HPDLF) and gingival (HGF) fibroblasts in culture. HPDLF and HGF were cultured from explants of healthy gingival tissue and freshly extracted teeth. Cultures of 8×10⁴ cells/ml were exposed to medium containing ³H-uridine and ³⁵S-methionine with TGF-β1 at concentrations from 10^-9 M to 10^-21 M, or control medium, for up to 60 hours in order to assess RNA and protein synthesis. Protein concentrations of comparable cultures were also assayed colorimetrically. Results were reported as specific activity (CPM/μMg protein). The results indicate that 10^-9 M TGF-β1 treated cultures showed a significant increase in RNA synthesis by HPDLF and HGF over time, as compared to the control cultures. HPDLF showed a significant increase in protein synthesis over time while that by HGF was not significant as compared to the control cultures. Lower concentrations of TGF-β1 demonstraled no significant differences from control. Results suggest that the effects of TGF-β1 on HPDLF and HGF are both time and dose dependent, with 10^-9 M TGF-β1 providing the best response of those concentrations tested. These findings support the concept that TGF-β1 may play a role in periodontal regeneration due to its ability to promote fibroblast RNA and protein synthesis. The results also demonstrate that although these two cells types appear morphologically similar, they exhibit distinct biological responses to growth factors such as TGF-β1.     

牙骨质基质提取物对两种细胞在牙根表面的细胞生物学研究

目的：观察牙骨质基质提取物能否促进牙周膜成纤维细胞、牙龈成纤维细胞向牙根表面移行、附着和趋向。方法：用细胞培养法和图像分析法分析细胞的附着和趋向。结果：发现牙骨质基质提取物能明显提高牙龈成纤维细胞和牙周膜成纤维细胞在牙根表面的附着，而且，随着培养时间的延长，牙骨质基质提取物促牙龈成纤维细胞和牙周膜成纤维细胞附着的功能更加显著。结论：牙骨质基质提取物可较好地促进牙龈成纤维细胞和牙周膜成纤维细胞在未脱矿的牙根表面上的附着、移行和趋向     

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.     

Effect of three commercial mouth rinses on cultured human gingival fibroblast: an in vitro study.

AIM: To examine the effect of three commercial mouth rinses (Hexidine 0.2%, Listerine Cool Mint, Betadine 1%) upon cultured human gingival fibroblast proliferation. MATERIALS AND METHODS: Human gingival fibroblasts were cultured and incubated in Dulbecco's Minimum Eagle's Medium containing Chlorhexidine, Listerine, Povidone-Iodine at varying concentrations (1%, 2%, 5%, 10%, 20% and 100% of the given solution) at 37 degrees C for 1, 5 and 15 min. Control cells received an equal volume of Dulbecco's Minimum Eagle's Medium without adding mouth rinses, for similar duration of exposure at 37 degrees C. Following incubation the media were removed, cells were washed twice with medium, supplemented with 10% Fetal Bovine Serum, and fibroblasts in the test and control group were allowed to recover in the same media for 24 h. RESULTS: In all the three groups, the proliferation inhibition was dependent on the concentration of solublized mouth rinses in the cell culture but independent of the duration of exposure to all three mouth rinses. The results showed that all three solutions were toxic to cultured human gingival fibroblasts, Chlorhexidine being the most cytotoxic. It was seen that at dilute concentrations (1% and 2% of given solutions) Listerine was more cytotoxic than Chlorhexidine and Povidone-Iodine. CONCLUSION: These results suggest that Chlorhexidine, Listerine and Povidone-Iodine are capable of inducing a dose-dependent reduction in cellular proliferation of fibroblasts. The results presented are interesting, but to know the clinical significance, further studies are needed.     

Influence of alloying elements on cellular response and in-vitro corrosion behavior of titanium-molybdenum-chromium alloys for implant materials

PurposeNot all elements with β-stabilizing properties in titanium alloys are suitable for biomaterial applications, because corrosion and wear processes release the alloying elements to the surrounding tissue. Chromium and molybdenum were selected as the alloying element in this work as to find balance between the strength and modulus of elasticity of β-titanium alloys. This study aimed to investigate the effect of Titanium-10Molybdenum-10Chromium (Ti-10Mo-10Cr), Titanium-10Chromium (Ti-10Cr) and Titanium-10Molybdenum (Ti-10Mo) on the elemental leachability in tissue culture environment and their effect on the viability of human gingival fibroblasts (HGFs).MethodsEach alloy was immersed in growth medium for 0–21 days, and the elution was analyzed to detect the released metals. The elution was further used as the treatment medium and exposed to seeded HGFs overnight. The HGFs were also cultured directly to the titanium alloy for 1, 3 and 7 days. Cell viability was then determined.ResultsSix metal elements were detected in the immersion of titanium alloys. Among these elements, molybdenum released from Ti-10Mo-10Cr had the highest concentration throughout the immersion period. Significant difference in the viability of fibroblast cells treated with growth medium containing metals and with direct exposure technique was not observed. The duration of immersion did not significantly affect cell viability. Nevertheless, cell viability was significantly affected after 1 and 7 days of exposure, when the cells were grown directly onto the alloy surfaces.ConclusionsWithin the limitation of this study, the newly developed β-titanium alloys are non-cytotoxic to human gingival fibroblasts.     

Cytotoxic effects of gingival retraction cords on human gingival fibroblasts in vitro

The objective of this study was to determine the cytocompatibility of three different extracts of gingival retraction cords and to compare the cytotoxic effect of these materials on human gingival fibroblasts. Gingival retraction cords impregnated with aluminium sulphate (Gingi-Aid), dl-adrenaline HCl (Gingi-Pak) and non-drug-impregnated cord (Gingi-Plain) were eluted with culture medium for 10 min and 24 h. Cytotoxicity was judged using a tetrazolium bromide reduction assay. Our data demonstrated that gingival retraction cords applied alone almost completely inhibited cell viability (P < 0.05). In addition, the results also showed that the eluates from aluminium sulphate-impregnated cord, dl-adrenaline HCl-impregnated cord and non-drug-impregnated cord were cytotoxic to primary human gingival fibroblast cultures (P < 0.05). The cell viability of incubation of gingival fibroblasts containing 10-min eluates of aluminium sulphate, dl-adrenaline HCl and non-drug-impregnated cord was 61, 21 and 70%, respectively. The cell viability of incubation of gingival fibroblasts containing 24 h eluates of aluminium sulphate, dl-adrenaline HCl and non-drug-impregnated cord was 68, 58 and 72%, respectively. It was found that dl-adrenaline HCl-impregnated gingival retraction cord was the most toxic gingival retraction cord among the materials tested in all cultures (P < 0.05). The cytotoxicity decreased in an order of dl-adrenaline HCl-impregnated cord > aluminium sulphate-impregnated cord > non-drug-impregnated cord. The extent or degree of the cytotoxicity depended on the materials tested. Gingival retraction cords have significant potential for gingival toxicity. Careful management of gingiva retraction cords would lower the risk of potential gingival tissue damage during clinical application procedure and thus increase the success of prosthodontic procedures.     

Regulation of human gingival fibroblast growth and synthetic activity by cyclosporine-A in vitro

Gingival overgrowth is an adverse side-effect seen in a proportion of patients taking cyclosporin-A which indicates that cyclosporine-A may modulate the activities of cells other than T lymphocytes. Therefore, the effect of cyclosporine on human gingival fibroblasts has been studied in vitro. Cyclosporine-A was found to stimulate DNA synthesis and the proliferative activity of these cells with maximal stimulation noted at a concentration of 10(-9) g/ml. Although this stimulation was most noticeable in the presence of 10% fetal calf serum, proliferation still occurred in serum-free medium. In the presence of lipopolysaccharide, at a concentration which normally inhibits gingival fibroblast proliferation, cyclosporine retained its capacity to stimulate proliferative activity. Fibroblasts isolated from overgrown gingival tissue responded to a greater extent than those isolated from a healthy site from the same individual. This stimulatory effect was not restricted to gingival fibroblasts, since human foreskin fibroblasts responded in a similar fashion. Cyclosporine-A did not significantly alter protein or proteoglycan production by these cells. These responses are considered to reflect the in vivo response of gingival overgrowth in patients taking cyclosporine-A. The reversal of lipopolysaccharide inhibition of gingival fibroblast proliferation by cyclosporine-A may explain, in part, why gingival overgrowth is most prominent in areas of heavy dental plaque accumulation.