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判别，传代培养时牙龄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龄成纤维细胞；碱性磷酸酶活性测定及染色法显示两者有明显的判别。结论：体外培养的两种细胞在形态及排列上相似，但百细胞亚型的组成上存在差异。     

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。     

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.     

两种方法培养人牙周膜成纤维细胞成功率的比较

目的探讨酶消化组织块法及传统组织块法培养人牙周膜成纤维细胞的成功率,并比较两种方法获得的细胞在形态学和生物学特性上的差异。方法分别采用酶消化组织块法及传统组织块法培养人牙周膜成纤维细胞各20份,观察细胞形态并通过免疫细胞化学染色法检测波形蛋白、角蛋白的表达情况,比较两种培养方法的成功率。结果酶消化组织块法成功率为65%,传统组织块法为30%,两者差异有统计学意义(P<0.05)。两组细胞均以梭形为主,波形丝蛋白染色阳性,角蛋白染色阴性,符合人牙周膜成纤维细胞的形态学特征和生物学特性。结论酶消化组织块法培养成功率高于传统组织块法。两种方法获得的细胞在形态学和生物学特性上无明显差异。     

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。     

人牙髓成纤维细胞等的体外培养、生长特性和鉴定

本实验从人恒牙分离牙髓组织,牙周组织和取鼠胚胎骨作体外培养。收集标本40个,培养出牙髓成纤维细胞7株,牙周膜成纤维细胞16个标本生长原代细胞8株,5个胚胎骨组织均生长7d以上。牙髓和牙周膜成纤维细胞形态典型,抗波形丝蛋白阳性。证明是中胚层来源的成纤维细胞,细胞生长曲线分裂指数显示细胞增殖正常,牙周膜细胞增长速度快于牙髓细胞。说明体外培养人牙髓和牙周膜细胞是可行的。     

人牙周膜成纤维细胞在纯钛表面附着的电镜观察

目的：研究人牙周膜成纤维细胞(PDLF)在纯钛金属表面的结合形式和超微结构。方法：将纯钛试件放在12孔培养板内，取生长良好的第五代人牙周膜成纤维细胞接种在试件表面，培养72h后取出，原位包埋法制作透射电镜标本，透射电镜观察。结果：人牙周膜成纤维细胞在纯钛表面附着形式不同于上皮细胞在金属表面的附着形式，细胞与金属结合界面中未观察到典型半桥粒结构。人牙周膜成纤维细胞胞浆中，细胞器发达，富含与蛋白质合成、代谢及增殖等生物学功能有关的细胞超微结构，如粗面内质网、线粒体、核糖体、细胞核等。结论：人牙周膜成纤维细胞在钛金属表面的附着形式，不同于上皮细胞的附着形式，表现为蛋白合成旺盛，可能为细胞直接附着，其具体附着形式还待进一步研究。     

两种体外培养人牙周韧带成纤维细胞方法比较

目的探索一种在体外短时间内简便、可靠获取大量人牙周韧带成纤维细胞(human periodontal ligament fibroblast,HPLF)、建立稳定的体外培养体系的方法。方法采用酶消化法和组织贴块法进行HPLF体外原代培养及传代培养的对比研究。通过细胞形态学、超微结构观察及波形蛋白和角蛋白免疫组化染色等对细胞进行定性研究;测定细胞生长曲线了解细胞生长基本规律及其增殖能力。结果采用酶消化法和组织贴块法均可成功的进行HPLF连续传代培养。最高传代数为30代。培养的细胞具有成纤维细胞的典型形态,波形蛋白染色阳性,角蛋白染色阴性,生长稳定期倍增时间为48~72h。组织块培养法需培养时间长,原代培养获取的细胞量较少,较难传代。酶消化法可短时间内获取大量细胞,细胞产量高,但操作手续复杂易污染,细胞易受损伤。结论成功建立了一个稳定的HPLF体外培养体系。除常用的组织块法外,胰蛋白酶及胶原酶联合消化法不失为一种简便、快速、可靠的组织原代分离培养方法。     

体外培养人牙周膜成纤维细胞及其成骨表型特征检测

目的　探讨牙周膜成纤维细胞作为牙周组织工程种子细胞的可行性。方法　采用酶消化法体外培养人牙周膜成纤维细胞 ,以人胎儿皮肤成纤维细胞为对照检测细胞碱性磷酸酶表达及矿化能力。结果　体外培养人牙周膜成纤维细胞与人胎儿皮肤成纤维细胞相比表达更高水平的碱性磷酸酶 (P <0 .0 5 ) ,矿化诱导液连续培养 30d均可观察到矿化结节形成。结论　采用酶消化法可快速简便地获取原代人牙周膜成纤维细胞。体外培养牙周膜成纤维细胞具有成骨样细胞表型特征 ,与人胎儿皮肤成纤维细胞相比更具分化潜能 ,可作为牙周组织工程种子细胞来源     

体外培养碱性成纤维细胞因子对人牙周膜成纤维细胞生物学特性的影响

目的：探讨体外培养过程中,碱性成纤维细胞生长因子（bFGF）对人牙周膜成纤维细胞（hPDLFs）生物学特性的影响。方法：体外分离培养人牙周膜成纤维细胞并鉴定,加入不同浓度bFGF（1ng／ml、10ng／ml、50ng／ml、100ng／ml）培养,MTT方法检测细胞增殖情况;并对细胞进行矿化诱导,检测细胞的碱性磷酸酶活性。结果：hPDLFs呈星形或长梭形,免疫组化波丝蛋白阳性,角蛋白阴性,证实该细胞来源可靠。bFGF在一定浓度范围内,与细胞增殖成正比,而在本实验培养条件下（10％FBS）bFGF最大效应浓度为10ng／ml。矿化诱导条件下,碱性磷酸酶活性明显增加,在联合应用bFGF的情况下,100ng／ml组碱性磷酸酶活性明显高于其他组。结论：不同浓度bFGF对人牙周膜成纤维细胞生物学行为的影响不同,在一定浓度条件下,低浓度bFGF促进人牙周膜成纤维细胞的增殖,而高浓度bFGF作用于人牙周膜成纤维细胞可能更易于分化。     

Human periodontal ligament in vitro: Cell culture passage effect on collagen gel contraction

Fibroblasts seeded within a lattice of a Type I collagen gel will exert tension on collagen fibrils and eventually contract the gel, forming a three-dimensional network. Using adult human tissue, the ability of PDL fibroblasts (HPDL) and human gingival fibroblasts (HGF) to contract collagen gels was compared. All cells were grown in monolayer and cell passage was performed as confluency was reached. Gel diameter was measured for a 10-d period after seeding the cells in the matrix. At d 1 through 10, the HGF contracted the gel to a greater extent than the HPDL. HPDL did not contract at a significantly different rate than fibroblasts derived from human sclera or human foreskin. When early passage HPDL and HGF were compared, the early passage HPDL cells contracted gels at a significantly reduced rate. The early passage HPDL cells demonstrated a large population of cells that did not display a normal extension of pseudopodia. However, the late passage HPDL gels contained a majority of cells displaying a normal cellular process extension, suggesting a heterogeneity of the cell population. This data supports the hypothesis that the cell culture monolayer environment may alter the functional characteristics of the HPDL, possibly by selecting against a subpopulation of cells which is present in primary culture and in vivo .     

Transwell滤膜上人牙周膜及牙龈成纤维细胞细胞层完整性的研究

目的 探讨生长在Transwell滤膜上人牙周膜成纤维细胞(human periodontal ligament fibroblasts,HPDLF)及牙龈成纤维细胞(human gingival fibroblasts,HGF)细胞层完整性,为研究牙周细胞对药物的跨细胞转运提供依据.方法 将原代培养的HPDLF及HGF分别接种到Transwell聚酯滤膜上,在1到4周内行跨膜电阻测定,连续以光学显微镜观察细胞的生长及排列情况,2周时对滤膜纵切面行光学显微镜及透射电子显微镜观察,并以渗漏标志物荧光素钠检测模型的渗漏.结果 HPDLF及HGF接种1周后细胞基本汇合,到2周时细胞连接紧密且完整,细胞呈复层生长,跨膜电阻分别达(56.14±7.43)及(57.34±7.62)Ω·cm~(-2),并持续到第4周;细胞接种2周后细胞药物转运模型经荧光素钠孵育30 min检测细胞旁渗漏小于1%.结论 HPDLF及HGF在Transwell滤膜上生长2周后细胞排列情况可模拟体内牙周膜和牙龈生理状态,且渗漏标志物的检测及跨膜电阻等结果显示细胞完整,旁渗漏很低,符合药物细胞转运模型的要求,可作为研究牙周细胞跨细胞转运药物的体外模型.     

人牙周膜细胞不同分离培养方法的比较研究

目的探讨组织块法、酶解组织块法和酶消化法体外分离培养人牙周膜细胞的优缺点。方法分别应用组织块法、酶解组织块法及酶消化法进行人牙周膜细胞分离培养,鉴定细胞来源,利用倒置显微镜观察细胞生长状况。结果应用组织块法从24块人牙周膜组织中成功分离培养出人牙周膜细胞,成功率96%（24/25）,细胞生长状态良好;应用酶解组织块法成功率80%（16/20）;应用酶消化法成功率75%（12/16）。结论组织块法原代培养人牙周膜细胞方法简单且成功率较高。     

SEM observations of the attachment of human periodontal ligament fibroblasts to non-demineralized dentin surface in vitro

PURPOSE: The purpose of this investigation was to study in-vitro the attachment behavior of human periodontal ligament (HPDL) fibroblasts to nondemineralized dentin surface using scanning electron microscope. STUDY DESIGN: Thirty root slices of freshly extracted human teeth of 4 mm thickness as well as six 5x5 mm glass slides used as a control were used in this study. The dentin surface of the root slices was not treated with any chemicals to remove the smear layer. The root slices and the glass slides were placed in tissue culture clusters and an amount of 1 ml of HPDL fibroblast cell suspension was placed over the dentin surface of the root slices and the glass slides. They were then placed into an incubator at 37 degrees C and 100% humidity for 4, 24, and 72 hours. At the end of the incubation, the cells were fixed with glutaraldehyde and examined microscopically. RESULTS: Different shapes of fully spread cells were seen. The cells were attached firmly to the dentin surface by the cytoplasmic extension of the lamellipodia and microvilli which were seen extending inside the opening of the dentinal tubules. CONCLUSION: It was concluded that the human dentin surface provided an excellent surface for attachment of periodontal ligament fibroblasts. In addition, the smear layer did not affect the cell attachment.     

Cytotoxicity of new resin-, calcium hydroxide- and silicone-based root canal sealers on fibroblasts derived from human gingiva and L929 cell lines

AIM: To assess ex vivo the cytotoxic effects of five new root canal sealers (RC Sealer, Epiphany, EndoREZ, GuttaFlow and Acroseal) and three existing products (AH Plus, RoekoSeal and Apexit) using primary human gingival fibroblasts (HGF) and a mouse fibroblast cell line, L929. METHODOLOGY: Eight samples of each sealer were fabricated in sterile cylindrical Teflon blocks, 4.4 mm diameter and 2 mm height and then divided into two groups, fresh and aged specimens. Extraction of fresh specimens was carried out after setting whilst aged specimens were placed in Petri dishes and kept in a humid chamber at 37 degrees C for 7 days before extraction in cell culture medium using the ratio 1.25 cm(2) mL(-1). Undiluted eluates were used for the dimethylthiazol diphenyltetrazolium bromide (MTT) assay with HGF and L-929. Morphology of HGF cells was also examined by an inverted microscope using undiluted eluates of the sealers. The results were analysed using a two-tailed t-test (alpha = 0.05) between groups. RESULTS: Resin-based (Epiphany and EndoREZ) and calcium hydroxide-based (Apexit and Acroseal) sealers were significantly more cytotoxic than other sealers (P<0.05). However, L929 cells were more sensitive to Apexit and EndoREZ than HGF cells. RC Sealer showed mild cytotoxicity to HGF at both setting times. AH Plus did not exert any cytotoxic effect to HGF and aged specimens appeared to induce cellular proliferation. RoekoSeal and GuttaFlow also demonstrated mild cytotoxicity. GuttaFlow was slightly more cytotoxic to both cultures, especially when tested fresh. CONCLUSIONS: Toxicity varied but RC Sealer and GuttaFlow were the least toxic new sealers.     

Effects of mechanical force on primary human fibroblasts derived from the gingiva and the periodontal ligament.

Previous experiments have shown that mechanical stress may alter the interactions between cells and extracellular matrix (ECM). The purpose of our study was to investigate the effects of mechanical load on metabolism and ECM expression of primary human periodontal cells. The influence of gravitational force on proliferation, lactate dehydrogenase (LDH) release, and tenascin expression of gingival (HGF) and periodontal ligament fibroblasts (HPDL), as well as their adhesion to various extracellular matrix (ECM) components, was determined. Cells were centrifuged in microplates or flat tubes for 16 hrs at 217 g. Neither an enhanced release of LDH nor an alteration of cell proliferation could be detected after centrifugation. However, the attachment of loaded gingival and periodontal ligament fibroblasts to all tested ECM components significantly decreased in comparison with controls (Wilcoxon-Mann-Whitney test; HGF, p < 0.05; HPDL, p < 0.01). Tenascin expression of mechanically stressed fibroblasts significantly increased in comparison with controls (p < 0.01).     

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.