血管内皮细胞对牙周组织细胞迁移的影响

目的: 通过血管内皮细胞与牙周组织细胞的复合培养模拟牙周组织再生环境, 研究血管内皮细胞对牙周组织细胞迁移的影响, 为明确血管内皮细胞在牙周组织再生过程中的作用奠定基础。方法: 应用原代培养的血管内皮细胞, 在Transwell嵌套中实现与人牙周膜成纤维细胞、牙龈成纤维细胞间的复合培养。分别以Transwell嵌套滤膜下腔面24 h的细胞数量检测垂直迁移能力, 以划痕实验后0、8、16和24 h计数划痕区域的细胞数量检测水平迁移能力, 以玻片试验及计算机辅助图像处理软件计算实验第1、4和7天细胞覆盖创面的面积检测覆盖创面能力, 并均与单独培养的2种牙周组织细胞作为对照。采用SPSS13.0软件包对实验结果进行统计学分析。结果: 血管内皮细胞存在时, 2种牙周细胞在垂直和水平方向的迁移量均显著高于单独培养组(P<0.01), 且牙周膜成纤维细胞垂直向的迁移量显著高于牙龈成纤维细胞(P<0.01), 但其水平迁移量在实验24 h时则显著低于牙龈成纤维细胞(P<0.01);在血管内皮细胞共存条件下, 2种牙周细胞创面覆盖能力在实验第7天高于对照组, 且牙周膜成纤维细胞的创面覆盖能力的增加显著高于牙龈成纤维细胞(P<0.01)。结论: 血管内皮细胞对牙周膜、牙龈成纤维细胞的迁移和创面覆盖有明显促进作用, 且血管内皮细胞对牙周膜成纤维细胞的垂直向迁移和创面覆盖能力的促进作用更为明显。     

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.     

FN对牙龈和牙周膜成纤维细胞生物学活性的影响

目的：观察纤维结合蛋白（FN）对牙龈成纤维细胞（GF）、牙周膜成纤维细胞（PDLF）的影响。方法：采用细胞培养、MTT比色测定、碱性磷酸酶（ALP）测定法、考马斯亮蓝法和茜素红染色法。结果：FN能促进两种细胞的增殖，对PDLF的碱性磷酸酶活性和蛋白合成也有促进作用，但对其矿化结节形成能力无显著影响。结论：FN能促进牙龈和牙周膜成纤维细胞的增殖及蛋白合成，但其促进细胞分化的能力有限，尚需其它生长因子的协助以促进组织再生。     

釉基质蛋白对牙周细胞覆盖体外创面的影响

目的：评价体外创面模型环境中,釉基质蛋白对牙周膜细胞、牙龈成纤维细胞覆盖创面能力的影响。方法：利用在盖玻片上形成的人牙周膜和牙龈成纤维细胞的融合培养物,以机械方法去除7mm宽的细胞层条带,形成体外创面模型。受损培养物在含有10％胎牛血清的培养液中继续培养2、6、9d,同时以100μg／ml的釉基质蛋白分别刺激牙周膜、牙龈成纤维细胞,并与不加釉基质蛋白者作对照。玻片经同定后作甲紫染色。以计算机辅助图像分析系统对细胞覆盖体外创面的面积进行百分比定量,采用SAS6、12软件包进行样本均数的t检验。结果：对照组组内牙周膜、牙龈成纤维细胞覆盖创面的面积存在差异,牙龈成纤维细胞覆盖创面面积在创面形成后第9天显著高于牙周膜细胞,差异有显著性（P〈0.05）。在加用100μg／ml釉基质蛋白的条件下,牙周膜细胞覆盖体外创面的面积较对照组明显增加．创面形成后第6、9天,牙周膜、牙龈成纤维细胞覆盖体外创面面积均无显著差异（P〉0．05）。结论：牙龈成纤维细胞覆盖创面的能力高于牙周膜细胞。釉基质蛋白有增进创面愈合,特别是牙周膜细胞创面覆盖的作用。     

壳聚糖/葡甘聚糖混合膜对人牙周膜细胞作用观察

目的:通过壳聚糖/葡甘聚糖膜单独、以及膜加载碱性成纤维细胞生长因子和第4代人牙周膜细胞共同培养,观察壳聚糖/葡甘聚糖膜对人牙周膜细胞增殖分化的影响。方法:观察不同质量比壳聚糖与葡甘聚糖共混膜对第4代细胞HPDLCs增殖影响及其对碱性磷酸酶活性(ALP)影响。结果:不同质量比的壳聚糖/葡甘聚糖混合膜对HPDLCs的增殖有促进作用,加载bFGF后,HPDLCs增殖得到增强,且壳聚糖/葡甘聚糖膜比3:0和1:2的混合膜还可增强ALP的活性水平。结论:壳聚糖/葡甘聚糖膜是一种潜在的牙周组织再生膜。     

一种牙周细胞体外创面愈合模型的构建

目的:构建一种可用于牙周组织创面愈合规律研究的体外创面愈合模型,比较牙周膜细胞和牙龈成纤维细胞覆盖体外创面的速度。方法:体外培养人牙周膜、牙龈成纤维细胞至玻片上各自形成单层融合。以机械方法刮除部分融合层细胞,构成25mm×7mm的无细胞区域,即牙周细胞体外创面。创面形成后继续于100mL/L胎牛血清环境下培养2、6、9d。玻片细胞固定后以结晶紫染色。以计算机辅助图像分析系统对创面特定区域采图,进行细胞覆盖面积定量和统计分析。结果:在所构建牙周细胞体外创面上牙龈成纤维细胞的覆盖速度高于牙周膜细胞。创面形成后第9天,牙龈成纤维细胞覆盖采图区域面积的百分比均数为62.26%,牙周膜细胞则为44.15%,差异有显著性(P<0.05)。结论:实验以细胞覆盖创面面积综合衡量细胞增殖和移动能力,在实验构建的牙周细胞体外创面模型上,牙龈成纤维细胞覆盖创面速度高于牙周膜细胞,能够体现两者在覆盖创面能力上的差异。实验构建的体外创面模型可用于牙周细胞创面愈合能力的研究,对探讨牙周组织创面愈合机制及其调控规律有所裨益。     

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。     

牙周膜成纤维细胞与三种可吸收引导组织再生膜生物相容性实验研究

目的：探讨牙周膜成纤维细胞与可吸收引导组织再生膜生物相容性，为牙周组织工程中支架材料选择提供依据。方法：将人牙周膜成纤维细胞与三种商品化引导组织再生膜：BME-10XR(中国医学科学院生物医学工程研究所)、BioMeshR(韩国Samyang公司)及Bio-GideR(美国Osteohealth公司)体外复合培养，进行形态学观察、细胞附着及增殖的检测。结果：人牙周膜成纤维细胞可在三种可吸收膜上附着、增殖，并复层生长。细胞在三种膜上的附着无显著性差异(P＞0．05)；在BMK-10XR及Bio-GideR上生长、繁殖明显好于BioMesh R(P＜0．05)。结论：BME-10X R及Bio-Gide R具有良好的生物相容性，有望成为牙周膜成纤维细胞的载体材料应用于牙周组织工程研究.     

组织工程方法构建复合培养细胞的引导组织再生膜

目的：探讨可吸收引导组织再生胶原膜（BME10－X）的生物相容性，构建引导组织再生膜。方法：用组织工程方法将人牙周膜成纤维细胞与BME10－X复合培养，培养第7、14d进行组织学及扫描电镜、透射电镜观察。结果：人牙周膜成纤维细胞可在BME10－X上贴附、增殖，分泌大量的细胞外基质，复层生长形成纤维组织结构，基本形成具有活性细胞及其基质的膜样组织，BME10－X在培养一周即出现可见的吸收。结论：BME10－X具有良好的生物相容性，可作为牙周膜细胞的载体应用于牙周组织工程研究。     

白细胞介素1对牙龈成纤维细胞增殖和前列腺素E_2产生的影响

本研究采用外用血单核细胞,以SiO_2作为刺激因子,经培养后,Sephadex G—75凝胶柱层析分离IL—1,观察IL—1对牙龈成纤维细胞PGE_2的产生和细胞增殖的影响,PGE_2的测定采用放免分析,以~3H—TdR掺入反映增殖情况。发现IL—1明显促进牙龈成纤维细胞PGE_2的产生,也明显促进牙龈成纤维细胞增殖,说明IL—1能通过促进PGE_2的产生而介导一系列牙周组织的病理过程,另外,还能通过促进牙龈成纤维细胞的增殖而在牙周组织修复中而起着重要作用。     

碱性成纤维细胞生长因子对成骨细胞和牙周膜成纤维细胞迁移、增殖的影响

目的　明确碱性成纤维细胞生长因子(bFGF)对体外成骨细胞和牙周膜成纤维细胞迁移、增殖的影响,以 探讨在牙种植体组织界面局部应用bFGF诱导类牙周膜形成的可行性。方法　同一只SD大鼠来源的成骨细胞和 牙周膜成纤维细胞经传代培养至第4代,建立体外创伤模型,分别在普通培养基和含bFGF的培养基中培养,观察 细胞迁移情况,四唑盐比色实验(MTT)测定细胞的增殖速度。结果　普通培养基中,成骨细胞迁移速度快于成纤维 细胞。加bFGF培养基中牙周膜成纤维细胞迁移速度明显快于其他各组,同时MTT结果显示加入bFGF能明显促进 两种细胞的增殖。结论　bFGF能明显促进牙周膜成纤维细胞的增殖、移行。     

Response of periodontal ligament fibroblasts and gingival fibroblasts to pulsating fluid flow: nitric oxide and prostaglandin E2 release and expression of tissue non-specific alkaline phosphatase activity

The capacity of the periodontal ligament to alter its structure and mass in response to mechanical loading has long been recognized. However, the mechanism by which periodontal cells can detect physical forces and respond to them is largely unknown. Besides transmission of forces via cell-matrix or cell-cell interactions, the strain-derived flow of interstitial fluid through the periodontal ligament may mechanically activate the periodontal cells, as well as ensure transport of cell signaling molecules, nutrients and waste products. Mechanosensory cells, such as endothelial and bone cells, are reported to respond to a flow of fluid with stimulated prostaglandin E2 (PGE2) and nitric oxide production. Therefore, we examined the PGE2 and nitric oxide response of human periodontal ligament and gingival fibroblasts to pulsating fluid flow and assessed the expression of tissue non-specific alkaline phosphatase activity. Periodontal ligament and gingival fibroblasts were subjected to a pulsating fluid flow (0.7 +/- 0.02 Pa, 5 Hz) for 60 min. PGE2 and nitric oxide concentrations were determined in the conditioned medium after 5, 10, 30 and 60 min of flowing. After fluid flow the cells were cultured for another 60 min without mechanical stress. Periodontal ligament fibroblasts, but not gingival fibroblasts, responded to fluid flow with significantly elevated release of nitric oxide and decreased expression of tissue non-specific alkaline phosphatase activity. In both periodontal ligament and gingival fibroblasts, PGE2 production was significantly increased after 60 min of flowing. Periodontal ligament fibroblasts, but not gingival fibroblasts, produced significantly higher levels of PGE2 during the postflow culture period. We conclude that human periodontal ligament fibroblasts are more responsive to pulsating fluid flow than gingival fibroblasts. The similarity of the early nitric oxide and PGE2 responses to fluid flow in periodontal fibroblasts with bone cells and endothelial cells suggests that these three cell types possess a similar sensor system for fluid shear stress.     

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。     

In vitro cell-type specific biological response of human periodontally related cells to platelet-rich plasma

OBJECTIVES: Platelet-rich plasma is a blood-derived fraction containing high concentrations of platelets and growth factors. Applied in the form of a gel on surgical wounds, it is able to stimulate hard and soft tissue repair and has been proposed for use in the field of periodontal regeneration. However to date, little is known about the biological interactions between platelet-rich plasma and periodontally related cells. In this study, we investigated the effects between platelet-rich plasma and cell populations involved in periodontal regeneration, namely primary human periodontal ligament cells, gingival fibroblasts and keratinocytes. MATERIAL AND METHODS: The proliferation of human periodontal ligament cells, gingival fibroblasts and keratinocytes by [3H]thymidine incorporation was assessed. The alkaline phosphatase activity and type I collagen levels of human periodontal ligament cells were also evaluated by a spectrophotometric assay and western blot analysis, respectively. RESULTS: Incubation of human periodontal ligament cells with platelet-rich plasma resulted in time-dependent growth stimulation (up to fourfold of control at 72 h). Likewise, an increase in the specific activity of alkaline phosphatase (fourfold at 6 days) and collagen (twofold at 7 days) was observed. Platelet-rich plasma also enhanced human gingival fibroblasts proliferation by twofold, whereas it inhibited human keratinocytes growth by 40%, with respect to their own controls at 72 h. CONCLUSION: Cell populations related to periodontal tissue were differently affected by platelet-rich plasma. In fact, a strong stimulation of human periodontal ligament cells proliferation, a minor increase in the growth rate of human gingival fibroblasts and a marked decrease of human keratinocytes proliferation were evident. In addition, in human periodontal ligament cells increased collagen and alkaline phosphatase activity levels were observed. These findings appear interesting in view of platelet-rich plasma utilization in periodontal regeneration.     

Emdogain对共培养成骨样细胞及内皮细胞成骨及成血管基因表达的影响

目的 评价emdogain对直接与间接共培养条件下成骨样细胞和内皮细胞成骨及成血管基因表达的影响以及其间的相关性。方法 将人脐静脉内皮细胞（HUVEC）和成骨样细胞（MG63）按2∶1比例于12孔板内进行直接及间接共培养,并使用不同浓度（0、0.1、1、10、50 μg/mL）的emdogain共培养细胞72 h。采用细胞计数仪结合流式细胞仪评价细胞增殖情况,并通过荧光激活流式细胞分选技术分选共培养细胞。对分选后的成骨样细胞及内皮细胞采用实时荧光定量PCR分别检测成骨相关基因碱性磷酸酶（ALP）、I型胶原蛋白（Col-1）及成血管相关基因血管内皮细胞生长因子（VEGF）、VEGF酪氨酸激酶受体1（Flt-1）、VEGF酪氨酸激酶受体2（KDR）、von Willebrand因子（vWF）、内皮细胞C蛋白受体（EPCR）、E选择素（E-Selectin）、促血管生成素2（Ang-2）的表达水平。采用SPSS24.0软件包对数据进行统计学处理。结果 在直接与间接共培养条件下,Emdogain对MG63及HUVEC细胞增殖的作用具有浓度依赖性。随着emdogain浓度增加,细胞增殖率逐渐下降。50 μg/mL浓度的emdogain对MG63及HUVEC细胞增殖抑制作用最为明显。随着emdogain浓度增加,MG63表面ALP和Col-1表达水平逐渐增加;50 μg/mL浓度条件下,表达水平最高且与其他组基因表达水平差异显著（P<0.01）。同一emdogain浓度条件下,直接共培养MG63表面ALP及Col-1表达水平均显著高于间接共培养（P<0.01）。除10 μg/mL组外,直接共培养条件下,VEGF表达水平均显著高于间接共培养（P<0.01）。50 μg/mL组直接共培养的HUVEC表面Flt-1、KDR、vWF、E-selectin基因表达水平显著高于间接共培养及直接共培养的其他各组（P<0.01）。间接共培养条件下,HUVEC表面EPCR及Ang-2基因表达水平显著高于直接共培养（P<0.05）。结论 Emdogain对共培养MG63和HUVEC增殖的作用具有浓度依赖性,50 μg/mL可能是emdogain作用的关键浓度。在直接共培养条件下,50 μg/mL emdogain抑制MG63及HUVEC细胞增殖,但能同时促进MG63及HUVEC细胞相关基因表达;且该条件下emdogain的作用显著强于其他实验条件,细胞间直接交联可能在其中发挥重要作用。     

雌激素对牙周膜细胞生物学性质的影响

目的：明确雌激素对体外牙周膜成纤维细胞迁移、增殖和碱性磷酸酶的影响，以探讨雌激素对牙周组织改建的影响。方法：SD大鼠来源的牙周膜成纤维细胞经传代培养至第四代，建立体外创伤模型，分别在普通培养基和含雌激素的培养基中培养，观察测量细胞迁移情况，运用MTT、酶联检测仪测定细胞的增殖速度，对细胞的ALP进行提取和测定。结果：MTT结果硅示加入雌激素对该细胞的增殖无明显影响；在含雌激素的培养基中成纤维细胞的迁移速度加快；同时牙周膜成纤维细胞的ALP表达娃著提高。结论：雌激素能明显促进牙周膜成纤维细胞的迁移，促进其分化。     

牙龈成纤维细胞、牙周膜细胞对白细胞介素8的趋化反应

目的评价牙龈成纤维细胞、牙周膜细胞对白细胞介素8（interleukin-8,IL-8）的趋化反应。方法将体外培养第6代的牙龈成纤维细胞和牙周膜细胞（2.5×10     

Synthesis of cementum-like tissue in vitro by cells cultured from bone: a light and electron microscope study

Cells obtained from calvariae of fetal rats, human gingival connective tissue and periodontal ligament of rat molars were each co-cultured in vitro with non-demineralized and partly demineralized root slices in nutrient medium containing 50 μGg/ml ascorbic acid and 10 mM B-glyeerophosphate, in order to determine whether the root slices could affect the phenotype expressed by the cells. The cultures were examined by light and transmission electron microscopy. Nodules of bone-like tissue, resembling those previously described by others, were observed in the multilayers of cells obtained from rat calvariae. but not in the multilayers of gingival and periodontal ligament cells, after 30 days of culture. Calvaria cells associated with the root slices produced tissues exhibiting ultrastructure that resembled that of bone or cellular cementum. acellular cemenlum and afibrillar cementum in vivo when examined after the same length of time. The tissues were never found in cultures of gingival fibroblasts or periodontal ligament cells. These observations suggest, first, that cells derived from bone can express in vitro the phenotype for the cementums; second, that cells obtained from human gingival and rat periodontal ligament do not do so when cultured under similar conditions; and third, the possibility that the osteoblasts, cementoblasts and their progenitors that are found in the periodontal ligament could, at least in part, have their origin and migrate there from the endosteal spaces of the alveolar process.