成纤维细胞生长因子对牙周膜细胞群及克隆钙化的作用

目的观察碱基成纤维细胞生长因子（ｂａｓｉｃｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒ，ｂＦＧＦ）对牙周膜（ｐｅｒｉｏｄｏｎｔａｌｌｉｇａｍｅｎｔ，ＰＤＬ）细胞克隆体外钙化的作用。方法对３颗前磨牙的牙周膜细胞进行细胞克隆化，观察ｂＦＧＦ对ＰＤＬ细胞克隆钙化能力的影响，测定细胞牙骨质附着蛋白（ｃｅｍｅｎｔｕｍｄｅｒｉｖｅｄａｔａｃｈｍｅｎｔｐｒｏｔｅｉｎ，ＣＡＰ）的结合力及碱性磷酸酶（ａｌｋａｌｉｎｅｐｈｏｓｐｈａｔａｓｅ，ＡＬＰ）的表达。结果ｂＦＧＦ使ＰＤＬ细胞克隆钙化率增加了１１％。ｂＦＧＦ依赖性钙化克隆与非ｂＦＧＦ依赖性钙化克隆相比具有较低的ＡＬＰ表达率和较高的ＣＡＰ结合力。结论ｂＦＧＦ可在体外促进ＰＤＬ细胞克隆钙化的形成，ｂＦＧＦ依赖与非依赖性钙化克隆是不同的细胞种群。     

人牙周膜细胞不同生物学特性的表达

目的：观察人牙周膜细胞生物学特性表达。方法：采用细胞克隆技术，以细胞的牙骨质附着蛋白（ Ｃ Ａ Ｐ） 亲和力、碱性磷酸酶（ Ａ Ｌ Ｐ） 表达率和细胞的钙化能力为指标，对５ 名患者的５ 个正常前磨牙的牙周膜（ Ｐ Ｄ Ｌ） 细胞进行了初步的鉴定。结果：在接种的６６９ 个克隆细胞中，仅有４３ ．６ ％ 的克隆细胞具有持续增殖潜力。在这些生长克隆细胞中，非钙化细胞所占比例要多于钙化细胞，但这两类细胞中分别存在着 Ａ Ｌ Ｐ 表达和 Ｃ Ａ Ｐ 亲和力有着不同表现的细胞。结论：牙周膜中存在有不同表现型的细胞。提示牙周组织再生是一复杂的过程。这些细胞在牙周组织再生中起着什么作用，尚待进一步研究。     

体外培养的SD大鼠类成牙骨质细胞生物学性状初步探讨

目的：对SD大鼠磨牙牙骨质的类成牙骨质细胞分泌碱性磷酸酶，骨钙素以及形成矿化结节的能力进行初步研究。方法：对12周龄SD大鼠第一磨牙类成牙骨质细胞进行骨钙素免疫组化染色和碱性磷酸酶活力测定，并利用茜素红S，检测在条件培养基下细胞形成钙化结节的情况；利用计算机图像分析仪对细胞骨钙素免疫组化染色切片进行光密度定量分析。结果：骨钙素在体外培养的类成牙骨质细胞中被强烈表达，而碱性磷酸酶在类成牙骨质细胞中几乎无活性。类成牙骨质细胞形成钙化结节茜素红S染色阳性。结论：体外培养的类成牙骨质细胞表达矿化相关蛋白，但几乎没有碱性磷酸酶活性，钙化条件下，能在体外形成钙化结节。     

Emdogain诱导人牙周膜细胞群向成牙骨质细胞分化的研究

目的探讨人牙周膜细胞群（human periodontal ligament cell populations,hPDLPs）在Emdogain（EMD,商品化的猪釉基质蛋白）诱导下生物学特性的改变。方法组织块法分离培养牙周膜细胞,用含100 mg/L EMD的培养液诱导6 d,光镜下观察细胞形态改变,扫描电镜观察细胞在牙骨质片上的形态变化。免疫细胞化学检测成牙骨质细胞相关矿化蛋白：骨涎蛋白（bone sialoprotein,BSP）、Ⅰ型胶原蛋白（collagenⅠ,COLⅠ）的表达。结果EMD诱导后细胞由长梭形向多角形的类成牙骨质细胞分化。免疫组织化学检测结果显示诱导后BSP、COLⅠ的表达增强。结论EMD可以诱导人牙周膜细胞群向类成牙骨质细胞方向分化。     

牙周病患牙根面的细胞附着和细胞扩展

目的：研究人牙周膜成纤维细胞(PDL)对牙周病患牙和健康牙牙根面的早期附着和扩展，以及脂多糖(LPS)对其的可能作用。方法：收集和制备22例牙周病患牙牙根组织切片，20例健康牙牙根组织切片。PDL取自健康的牙周膜。将培养的PDL接种于牙根组织切片，孵育lh37℃，作细胞计数，检测细胞的早期附着；接种培养的PDL于牙根组织切片，孵育48h37℃检测细胞扩展。用血细胞计数器计数扩展细胞。用涂布LPS的组织培养孔试验LPS对细胞附着、细胞扩展的影响。结果：人牙周膜成纤维细胞在牙周病患牙和健康牙牙根表面的早期附着没有差异，而两者间细胞的扩展有显著差异，健康牙高于患牙。患牙根面的LPS可能是抑制根面细胞扩展的因素之一。结论：牙周病患牙和健康牙的根面均可发生PDL的早期附着，但随着时间的推移，患牙根面的细胞不能发生扩展。     

人牙周膜细胞中不同细胞表现型的表达

本项实验利用细胞克隆技术，以细胞的牙骨质附着蛋白（ｃｅｍｅｎｔｕｍｄｅｒｉｖｅｄａｔａｃｈｍｅｎｔｐｒｏｔｅｉｎ，ＣＡＰ）结合力、碱性磷酸酶（ＡＬＰ）表达率和细胞体外钙化能力为指标，对人牙周膜细胞进行了克隆化和分类。１材料与方法：采用稀释法对５颗...     

一种蛋白酶体阻断剂对牙周膜细胞生物学性能的影响

目的探讨一种蛋白酶体阻断剂bortezomib对牙周膜（periodontal ligament,PDL）细胞生物学性能的影响。方法小鼠牙周膜细胞系（mouse PDL clone,MPDL22）在细胞培养矿化液中加入bortezomib。免疫印迹法分析bortezomib对MPDL22细胞泛素-蛋白酶体系统（ubiquitin-proteasome pathway,UPP）的阻断作用。分别应用台盼蓝染色和细胞增殖试剂盒检测bortezomib对MPDL22细胞活性和增殖的影响,碱性磷酸酶染色和茜素红染色检测bortezomib对MPDL22细胞的碱性磷酸酶活性和矿化结节形成的影响。结果 Bortezomib以浓度依赖性方式抑制MPDL22细胞的UPP,对MPDL22细胞活性和增殖无明显影响（P〉0.05）。Bortezomib提高了MPDL22细胞的碱性磷酸酶活性（P〈0.001）,促进MPDL22细胞形成矿化结节。结论 Bortezomib可阻断MPDL22细胞的蛋白酶体途径,提高碱性磷酸酶活性,促进矿化结节形成,提示bortezomib有可能应用于牙周组织再生治疗。     

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     

年龄对牙周膜细胞钙化表达的影响

目的探讨不同年龄人的牙周膜（ｐｅｒｉｏｄｏｎｔａｌｌｉｇａｍｅｎｔ，ＰＤＬ）细胞参与牙周组织再生的能力和影响因素。方法对青年人（４颗牙）和老年人（３颗牙）的正常牙周膜细胞进行细胞克隆化，观察碱基成纤维细胞生长因子（ｂａｓｉｃｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒ，ｂＦＧＦ）对ＰＤＬ细胞克隆体外钙化能力的影响。结果青年组钙化细胞约占接种细胞数的１７７％，而老年组仅占４１％。可见老年人ＰＤＬ细胞的钙化能力明显低于青年人。当ｂＦＧＦ作用后，青年组所获钙化细胞克隆数较前增加２６３％；而老年组增加３４％。结论老年组ＰＤＬ中前体细胞的数量和对ｂＦＧＦ的生物学应答均低于青年组。对老年人牙周组织再生治疗原则和研究方向应有别于青年人，应着重于增加老年人ＰＤＬ中干细胞池的数量和激活前体细胞的生物学功能。     

牙骨质基质提取物促进牙周结缔组织细胞在牙根表面上增？…

目的：证实牙骨质基质提取物可是牙周结缔组织细胞在牙根表面上增殖。方法：用细胞培养法和增殖细胞记数法。结果：加入牙骨质基质提取物的实验组无认是牙龈成纤维细胞还是牙周膜细胞的增殖能力均有显著的提高。结论：牙骨质基质提取物可促进牙龈成纤维细胞和牙周膜成纤维细胞在牙根表面上的增殖，牙骨质基质提取物对牙龈成纤维细胞的促有丝分裂作用强于对牙周膜成纤维细胞的作用。     

N-cadherin expression during periodontal ligament cell differentiation in vitro.

BACKGROUND: When confluent periodontal ligament (PDL) cells were cultured in the presence of dexamethasone (Dex), ascorbic acid (AA), and beta-glycerophosphate (GP), they underwent sequential differentiation, demonstrating distinct morphological characteristics. At 1 week, localized cell proliferation led to the formation of multilayers of cells. As cell differentiation progressed, they formed nodules by deposition of matrix in the clusters of cells at 2 weeks, and mineralized the nodules at 3 weeks. These changes implicate extensive cell-to-cell interactions. Cadherins are known to play an important role in establishing cell contacts during tissue formation. METHODS: To determine whether cadherins are involved in PDL cell differentiation, and the formation and mineralization of nodules by the cells in vitro, we investigated the expression of N-cadherin using immunofluorescence labeling and Northern blot analysis. RESULTS: Immunolabeling showed that N-cadherin was expressed in PDL cells in the stages of nodule formation and mineralization. Northern blot analysis demonstrated a 3-fold increase in the expression of N-cadherin mRNA in the stages. However, neither E-cadherin nor P-cadherin was expressed. CONCLUSIONS: Our data suggest that N-cadherin may play an important role in PDL cell differentiation and the formation of mineralized nodules by PDL cells.     

Employing a transgenic animal model to obtain cementoblasts in vitro

BACKGROUND: Proper formation of cementum, a mineralized tissue lining the tooth root surface, is required for development of a functional periodontal ligament. Further, the presence of healthy cementum is considered to be an important criterion for predictable restoration of periodontal tissues lost as a consequence of disease. Despite the significance of cementum to general oral health, the mechanisms controlling development and regeneration of this tissue are not well understood and research has been hampered by the lack of adequate in vitro experimental models. METHODS: In an effort to establish cementoblast cell populations, without the trappings of a heterogeneous population containing periodontal ligament (PDL) cells, cells were obtained from the root surface of first mandibular molars of OC-TAg transgenic mice. These mice contain the SV40 large T-antigen (TAg) under control of the osteocalcin (OC) promoter. Therefore, only cells that express OC also express TAg and are immortalized in vitro. Based on results of prior in situ studies, OC is expressed by cementoblasts during root development, but not by cells within the PDL. Consequently, when populations are isolated from developing molars using collagenase/trypsin digestion, only cementoblasts, not PDL cells, are immortalized and thus, will survive in culture. RESULTS: The resulting immortalized cementoblast population (OC/CM) expressed bone sialoprotein (BSP), osteopontin (OPN), and OC, markers selective to cells lining the root surface. These cells also expressed type I and XII collagen and type I PTH/PTHrP receptor (PTH1R). In addition to expression of genes associated with cementoblasts, OC/CM cells promoted mineral nodule formation and exhibited a PTHrP mediated cAMP response. CONCLUSIONS: This approach for establishing cementoblasts in vitro provides a model to study cementogenesis as required to enhance our knowledge of the mechanisms controlling development, maintenance, and regeneration of periodontal tissues.     

Periodontal ligament cell sheet promotes periodontal regeneration in athymic rats

Aim: The primary goal of periodontal treatment is regeneration of the periodontium. Current theories suggest that the periodontal ligament (PDL) cells have the capacity to participate in restoring connective and mineralized tissues, when appropriately triggered. We evaluated whether human PDL cell sheets could reconstruct periodontal tissue. Material and Methods: To obtain the cell sheet, human PDL cells were cultured on temperature‐responsive culture dishes with or without osteogenic differentiation medium. The cell sheets were transplanted on periodontal fenestration defects of immunodeficient rats. Forty rats were divided in two groups: in one group, cell sheets cultured with control medium were transplanted and in the other, cell sheets cultured with osteogenic differentiation medium were transplanted. The defects were analysed histologically and histomorphologically after healing. Results: Most of the experimental group exhibited a new cementum‐like layer and new attachment of collagen fibres to the layer. Histomorphological analyses indicated significant periodontal regeneration. The control group revealed dense extracellular matrix and fibre formation, but an obvious cementum layer was not observed. Conclusions: Transplanted PDL cell sheets cultured with osteogenic differentiation medium induced periodontal regeneration containing an obvious cementum layer and Sharpey's fibres. Thus, the method could be feasible as a new therapeutic approach for periodontal regeneration.     

Periodontal ligament cells from insulin-dependent diabetics exhibit altered alkaline phosphatase activity in response to growth factors.

BACKGROUND: Insulin-dependent or Type 1 diabetes mellitus (IDDM) has been associated with an increased severity of periodontal disease. Because periodontal ligament (PDL) cells play a significant role in maintenance and regeneration of mineralized tissue, the success of procedures, such as guided tissue regeneration, is directly related to the ability of these cells to augment mineralized tissue. The objective of this study was to examine the ability of PDL cells from long-standing IDDM patients to form mineralized tissue and to determine whether these cells would exhibit altered responses to exogenously added growth factors. METHODS: PDL cells were isolated from 4 patients with IDDM treated with insulin for at least 5 years and from systemically healthy donors. The cell isolates were tested for their ability to form mineralized nodules in vitro and to express alterations in alkaline phosphatase activity in response to exogenously added growth factors (transforming growth factor-beta (TGF-beta), platelet-derived growth factor-BB (PDGF-BB), and insulin-like growth factor-1 (IGF-1). Alkaline phosphatase activity was determined spectrophotometrically. RESULTS: Although all PDL cell isolates formed mineralized nodules in vitro, PDL cells from diabetics formed mineralized nodules more slowly than did the controls. Alkaline phosphatase activity was not altered by exposure of diabetic PDL cells to TGF-beta for 9 days. In contrast, non-diabetic isolates exhibited increased levels of activity with increasing concentrations, from 0.5 to 1.0 ng/ml. Alkaline phosphatase activity was significantly higher in non-diabetic, but not in diabetic, cell isolates exposed to TGF-beta at 1.0 ng/ml, when compared to non-treated controls. Diabetic cell isolates exhibited significantly lower alkaline phosphatase activity than the non-diabetic isolates when exposed to either TGF-beta, PDGF-BB, IGF-1 or a combination of PDGF-BB and IGF-1. CONCLUSIONS: These results suggest that the populations of PDL cells in insulin-dependent diabetics may be altered in their ability to form mineralized tissue and to respond to growth factors, functions affecting the maintenance and regeneration of the periodontium.     

不同代数人牙周膜细胞成骨特征稳定性的实验研究

目的：观察培养代数牙周膜细胞的成骨细胞表型特征的影响。方法：利用4、6、8、10代细胞进行以形态学、细胞生长曲线、碱性磷酸酶（ALP）活性测定以及矿化能力和面积的观察。结果：在细胞形态和超微结构、细胞生长曲线、碱性磷酸酶活性和矿化能力方面，第4、6、8、10代无明显差别；10代以后的细胞各方面指标减弱，提示细胞进入衰老期。结论：利用10代以内牙周膜细胞进行实验或移植其结果可靠。     

Gene profile in periodontal ligament cells and clones with enamel matrix proteins derivative

AIM: Evaluate enamel matrix proteins derivative effect on gene expression profiles in cultured human periodontal ligament cell population and its clones. MATERIAL AND METHODS: Human periodontal ligament (PDL) cells were explanted. Cell cloning was performed and clones classified into fibroblastic (FB) and mineralized tissue forming (MTF) according to their capacity to express alkaline phosphatase and form mineralized tissue. All cell cultures were grown for 7 days, with and without enamel proteins added to the medium. Following RNA extraction, expression profiling was performed by hybridization with a DNA micro-array. Selected genes differed from the control at a significant level smaller than p<0.01. RESULTS: Enamel proteins induced major qualitative changes in mRNA expression in all PDL cell populations, differently affecting the entire PDL cell population and its clones. In the entire PDL cell population, enamel proteins significantly enhanced PDL cell function, with a general effect on enhanced cell functional metabolism. CONCLUSIONS: Enamel proteins enhanced gene expression responsible for protein and mineralized tissue synthesis in the entire PDL population. In the MTF clones, nucleic acid metabolism, protein metabolism and signal transduction related genes were up-regulated, while in the FB clones, up-regulated genes were related to cell adhesion, nucleic acid metabolism and signal transduction.