Phenotypic comparison of periodontal ligament cells in vivo and in vitro

The mammalian periodontal ligament contains heterogeneous populations of connective tissue cells, the precise function of which is poorly understood. Despite close proximity to bone and the application of high amplitude physical forces, cells in the periodontal ligament (PL) are capable of expressing regulatory factors that maintain PL width during adult life. The study of PL homeostasis and PL cell differentiation requires culture and phenotypic methods for precise characterization of PL cell populations, in particular those cells with an inherently osteogenic program. Currently it is unknown if cells cultured from the PL are phenotypically similar to the parental cells that are present in the tissues. We have compared the phenotype of cells in vivo with cells derived from the PL and expanded in vitro to assess the general validity of in vitro models for the study of phenotypic regulation in vivo. Rat PL cells were isolated by either scraping the root of the extracted first mandibular molars (Group A), or by scraping the alveolar socket following extraction of first mandibular molars (Group B), or by obtaining a mixture of cells after disaggregating a block of tissue consisting of first mandibular molar, PL and the surrounding alveolar bone (Group C). Cultured cells at confluence were fixed and immunostained for alpha-smooth muscle actin (alpha-SMA), osteopontin (OPN), alkaline phosphatase (AP), or bone sialoprotein (BSP). For in vivo assessments, frontal sections of rat first mandibular molar were immunostained for alpha-SMA, OPN, AP and BSP. We examined osteogenic differentiation of cultured PL cell cultures by bone nodule-forming assays. In vivo and at all examined sites, > 68% of PL cells were immunostained for AP; approximately 50% and approximately 51% for OPN and alpha-SMA (p = 0.3), respectively, while only approximately 8% were positively stained for BSP (p < 0.01). Analysis of cultured PL cells in Groups A, B and C showed 54%, 53%, and 56% positive staining for alpha-SMA respectively; 51%, 56%, 54% for OPN; 66%, 70%, 69% for AP and 2.2%, 1.4% and 2.8% for BSP. The mean percentage of PL cells in situ stained for the different markers was similar to that of cultured PL cells (Group A approximately Group B approximately Group C in situ for p > 0.2) except for BSP which was 3 to 4 fold higher in vitro (p < 0.01). PL cell cultures treated with dexamethasone showed mineralized tissue formation for all groups (A, B, C), but no mineralized tissue formation was detected in the absence of dexamethasone. As PL cells express quantitatively similar phenotypes in vitro and in vivo, we conclude that the in vitro models used here for assessment of PL cell differentiation appear to be appropriate and are independent of the cell sampling method. Further, dexamethasone-dependent progenitors are present both on the root and bone-related sides of the PL.     

SOST基因在人牙周膜细胞矿化诱导过程中的表达

目的:探讨SOST基因在人牙周膜细胞矿化诱导过程中的表达变化,为进一步研究SOST基因在牙周组织中的作用提供理论基础。方法:在体外培养条件下,将矿化诱导液作用于人牙周膜细胞(7、14、21d),检测细胞矿化能力、SOST基因以及成骨标志基因的表达变化。采用SPSS 17.0软件包对数据进行统计学分析。结果:随着诱导时间的增加,人牙周膜细胞的碱性磷酸酶染色及矿化钙结节染色程度增加,成骨标志基因以及SOST基因的表达量在矿化诱导第14天及21天后明显升高,差异均有统计学意义(P<0.05),并具有时间依赖性。结论:牙周膜细胞在mRNA水平能够表达SOST,而且矿化诱导作用对人牙周膜细胞中SOST基因的表达有促进作用。提示SOST参与人牙周膜细胞的成骨分化过程,并对牙周组织改建有重要作用。     

Cellular responses of rat periodontal ligament cells under hypoxia and re-oxygenation conditions in vitro

Background and Objective:  The aim of this study was to investigate the responses of periodontal ligament cells under hypoxia and re-oxygenation conditions in vitro .
Material and Methods:  Periodontal ligament fibroblasts were isolated from rat incisors. In the hypoxia group, cells were incubated in 2% O₂ for 1–3 d. In the re-oxygenation group, cells were first incubated under the same conditions as the hypoxia group for 24 h and then were returned to normoxic conditions and cultured for 1–2 additional days.
Results:  Proliferation ratios increased in all groups in a time-dependent manner. Proliferation ratios in both the hypoxia and re-oxygenation groups were significantly higher than in the control group on days 2 and 3. Alkaline phosphatase activity was significantly higher in the hypoxia group than in the control and the re-oxygenation groups. The expression of bone sialoprotein mRNA was significantly higher in the hypoxia group than in the control group on days 1 and 2. The expression of vascular endothelial growth factor mRNA was significantly higher in the hypoxia group than in the control group on days 1 and 2. In the re-oxygenation group, the level of expression of bone sialoprotein mRNA and vascular endothelial growth factor mRNA were similar to those of the control group. The expression of heat shock protein 70 mRNA in the hypoxia group was similar to that in the control group, whereas in the re-oxygenation group it was statistically higher than in the other groups.
Conclusion:  These results suggest that periodontal ligament cells maintain their osteogenic ability in hypoxia and re-oxygenation conditions in vitro .     

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。     

改良法分离培养人牙周膜干细胞

目的 改良法体外分离培养人牙周膜干细胞(PDLSC),并进行鉴定.方法采用酶消化组织块法获得人牙周膜细胞,通过有限稀释法克隆化培养、分离得到PDLSC,用含10%FBS的α-MEM培养液培养并传代:测定克隆形成率:免疫组织化学检测角蛋白及波形蛋白表达:流式细胞术分析细胞周期及表面标志物STRO-1、CD146的表达;并...     

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.     

人牙周膜成纤维细胞在无血清培养液中的生长特性

目的：探讨人牙周膜成纤维细胞在无血清培养液中的生长特性。方法：用倒置显微镜和MTT法观察人牙周膜成纤维细胞在无血清培养液中的生长和增殖变化。结果：人牙周膜成纤维细胞在无血清培养条件下可以生长的增殖，但与含血清培养液相比，其增殖速率变缓，分化明显。结论：无血清培养液可应用于人牙周膜成纤维细胞的体外培养和实验研究中。     

酶消化贴壁组织块反复消化法培养人牙周膜细胞

目的：牙周膜细胞原代培养成功率较低,探讨经济、高效的原代人牙周膜细胞培养方法,提高原代培养成功率及获得大量原代细胞一直是近年来牙周细胞生物学的研究热点之一.方法：无菌刮出因正畸需拔出牙的牙周韧带,剪碎,用改良的酶消化-贴壁组织块-反复消化法培养细胞,MTT法检测其生长曲线,免疫组织化学鉴定波形蛋白与角蛋白,通过相差显微镜观察、骨分化诱导液诱导后用骨钙素免疫荧光染色、碱性磷酸酶染色和钙结节染色方法对所获得的细胞进行鉴定.结果：所培养的细胞表达波形丝蛋白,骨分化诱导后碱性磷酸酶染色、骨钙素染色和钙结节染色均呈阳性.结论：改良的酶消化-贴壁组织块-反复消化法可获得大量的人牙周膜细胞.     

In vitro effect of platelet-derived growth factor-BB on collagen synthesis and proliferation of human periodontal ligament cells

OBJECTIVES: Platelet-derived growth factor (PDGF)-BB is a polypeptide growth factor which has been shown to stimulate periodontal regeneration. In this study, we investigated the time- and dose-dependent effect of PDGF-BB on the proliferation and collagen synthesis of human periodontal ligament (PDL) cells. MATERIALS AND METHODS: For the proliferation assay, PDL cells were cultured in 0.01-10 ng ml(-1) of PDGF-BB for 12 or 24 h, and cell numbers were counted. For the collagen synthesis assay, PDL cells were cultured in 0.1-10 ng ml(-1) of PDGF-BB for 1 to 24 h. The ratio of collagen content in total protein was evaluated, and the gene expression of type I collagen was assessed quantitatively by Northern blotting analysis. RESULT AND CONCLUSIONS: PDGF-BB stimulated the proliferation of PDL cells in a time- and dose-dependent manner with the maximum effect at 10 ng ml(-1). PDGF-BB induced the collagen synthesis of PDL cells with the maximum effect for 24-h treatment, and 1 ng ml(-1) of PDGF-BB. PDGF-BB exhibits an inverse dose-dependent effect on proliferation and collagen synthesis by PDL cells. These findings suggest that PDGF-BB is one of the important regulators of the maintenance of the extracellular matrix in PDL, and may play an important role in the regeneration of PDL.     

人牙周膜体外分离培养及其成骨表型的研究

目的：体外培养人牙周膜细胞（human periodontal ligament fibroblasts hPDLFs）,并对其成骨表型特征进行研究。方法：采用胶原酶消化法获得大量成活率高的人牙周膜细胞。在DMEM培养基中进行原代及传代培养,免疫组化方法检测细胞波形丝蛋白及角蛋白的表达,鉴定细胞。使用矿化液诱导,然后应用碱性磷酸酶活性检测、Von Kossa染色等方法观察hPDLFs在矿化液作用下生物学特性的改变。结果：hPDLFs呈星形或长梭形,免疫组化波形丝蛋白阳性,角蛋白阴性,证实该细胞来源可靠。矿化诱导5d后,有部分hPDLFS转化为成骨样细胞,碱性磷酸酶活性显著升高,诱导20d后可见矿化结节形成。结论：酶消化法可以快速得到大量hPDLFs,且hPDLFs具有一定的成骨表型特征。     

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。     

聚己内酯电纺纤维支架培养人牙周膜细胞的体外研究

目的：研究聚己内酯（PCL）电纺纤维支架上人牙周膜细胞的生物学行为，初步探讨PCL电纺纤维支架材料应用于牙齿再生和牙周组织工程研究的可行性。方法：采用静电纺丝法制备PCL电纺纤维支架。组织块法培养人牙周膜细胞（PDLC），传代扩增后接种于PCL电纺纤维支架上。激光共聚焦显微镜、扫描电镜观察人PDLC在支架材料上的黏附、生长情况，MTT法检测PCL电纺纤维支架对PDLC增殖的影响。结果：PCL电纺纤维支架呈无纺多孔网状结构，直径范围为338～424nm，纤维形态光滑均一。激光共聚焦显微镜、扫描电镜显示PDLC在支架上贴附牢固，伸展充分，增殖旺盛并分泌大量的细胞外基质。MTT法检测显示：PCL电纺纤维支架材料对PDLC生长增殖的影响与对照组培养板相比无统计学差异（P〉0．05）。结论：PCL电纺纤维支架具有良好的生物相容性，有望作为一种新型的支架材料应用于牙周组织工程。     

体外培养人牙周膜细胞成骨基因表达谱的检测

目的:探讨体外培养人牙周膜细胞(human periodontal ligament cells,hPDL)成骨相关基因的表达谱,明确体外培养人牙周膜细胞的成骨潜能。方法:消化离心法收集体外培养人牙周膜细胞,提取总RNA,使用Super-array公司的点样数96点的人骨再生基因表达谱芯片检测人牙周膜细胞成骨相关基因的表达。结果:体外培养人牙周膜细胞有15种成骨相关基因无表达,81种基因有表达,其中表达较高的基因22种。结论:体外培养人牙周膜细胞具有部分成骨细胞基因表型特征,但成熟成骨细胞的特征基因表达量较低,提示人牙周膜细胞是具有成骨细胞分化潜能的成纤维细胞。     

人牙周膜细胞群多向分化潜能的实验研究

目的研究体外培养的人牙周膜细胞群（hPDLP）向成骨和成脂方向分化的潜能,为牙周组织工程提供可靠的种子细胞来源。方法组织块法分离培养人牙周膜细胞群,流式细胞术检测间充质干细胞标记CD146和STRO-1的表达;利用茜素红、油红O染色、免疫组化以及反转录聚合酶链反应（RT-PCR）等检测hPDLP的多向分化标志。结果第1代hPDLP的CD146和STRO-1阳性率分别是27.20%±3.98%和4.23%±4.08%;经矿化诱导可以形成矿化结节,有钙盐沉积;经成脂诱导可见特异性脂滴形成,特异性转录因子过氧化物酶体激活物增生受体2（PPARγ2）和脂蛋白脂酶（LPL）表达上调。第8代hPDLP相对第1代的矿化能力没有差异,但成脂方向分化潜能减弱。结论体外培养的hPDLP具有向成骨和成脂样细胞分化潜能,第1~3代细胞群明显具有牙周膜干细胞的多向分化潜能优势。     

不同培养条件下牙周韧带细胞矿化能力的比较研究

目的：探讨牙周韧带细胞体外常规状态及矿化诱导下钙化特性的差异。方法：用组织块培养法进行人牙周韧带细胞的原代培养，取第4代细胞用于实验，在体外长期培养，条件培养组加入矿化诱导液，常规培养组不加任何矿化诱导因素，倒置显微镜下观察矿化情况，茜素红与Von-Kossa染色显示钙盐沉积。结果：牙周韧带细胞在体外长期培养过程中，两组均表现为融合期、复层期、结节期、矿化期，形成肉眼可见的白色结节，茜素红与Von-Kossa染色均显示结节内有钙盐沉积。但常规培养组矿化所需的时间要比条件培养组长1周左右。结论：牙周韧带细胞在体外长期培养过程中，无论有无矿化诱导因素存在，均具有向矿化组织形成细胞分化的趋势，但矿化诱导因素的存在可以促进矿化结节的早期形成。     

Aging affects the phenotypic characteristics of human periodontal ligament cells and the cellular response to hormonal stimulation in vitro

Lossdörfer S, Kraus D, Jäger A. Aging affects the phenotypic characteristics of human periodontal ligament cells and the cellular response to hormonal stimulation in vitro. J Periodont Res 2010; 45: 764–771. © 2010 John Wiley & Sons A/S Background and Objective: Aging modulates the proliferative activity and organic matrix production of cells in vivo and in vitro. Here, we explore how aging affects the phenotypic characteristics of human periodontal ligament cells and their response to hormonal stimulation. Material and Methods: Fifth passage periodontal ligament cells from subjects aged 12–14 (group 1), 41–55 (group 2) and 61–70 years (group 3) were characterized for the expression of mesenchymal marker genes and proteins by real‐time PCR and flow cytometry. Confluent cultures were exposed to 10^?12 m parathyroid hormone(1–34) [PTH(1–34)] intermittently for three cycles. At harvest, cell number, alkaline phosphatase activity and osteocalcin production were determined by cell count, biochemical assay and ELISA. Results: The characterization of the cells revealed a decreased expression of osteoblast‐specific marker genes along with a lower percentage of cells presenting the respective proteins with age. An intermittent exposure of the cultures to 10^?12 m PTH(1–34) induced an increase of the cell number as opposed to a significant decrease of alkaline phosphatase activity and osteocalcin production. The cellular response to PTH(1–34) was strongest in group 1. Basal osteoprotegerin levels were highest in the cultures from the oldest donors and inhibited by intermittent PTH(1–34) in all groups. Conclusion: Our data indicate that periodontal ligament cells from older subjects display a less differentiated phenotype and a reduced response to intermittent PTH, suggesting a compromised ability to maintain tissue homeostasis and a limited possibility to support periodontal repair processes with age. The high basal osteoprotegerin expression in older subjects might serve as a compensatory mechanism.     

牙周膜干细胞的研究进展

牙周膜干细胞可分化为成骨细胞或成牙骨质细胞、脂肪细胞和胶原形成细胞,是牙周组织工程中新的种子细胞。笔者就牙周膜干细胞的生物学特性、牙周膜干细胞的多向分化潜能、牙周膜干细胞的移植及其潜在的临床应用价值作一综述。     

FHL2蛋白在人牙周膜细胞体外矿化过程中的表达

目的 探讨胞内信号转导分子FHL2蛋白在人牙周膜细胞(hPDLCs)体外矿化过程中的表达.方法 体外培养hPDLCs,实验组用矿化诱导液培养,对照组不加诱导液.培养0、14、28 d后,茜素红染色检测矿化结节的形成;免疫细胞化学法检测hPDLcs矿化诱导0、14d时FHL2蛋白的表达;同时采用半定量RT-PCR方法检测...     

淫羊藿苷对人牙周膜细胞增殖和骨保护素mRNA表达的影响

目的：研究淫羊藿苷对人牙周膜细胞（periodontal ligament cells，PDLCs）增殖和骨保护素（osteoprotegerin，OPG）mRNA表达的影响。方法：体外培养人PDLCs，用MTT法检测不同浓度的淫羊藿苷（0．001、0．01、0、1μg／mL）、不同时间（24、48、72、96h）作用下人PDLCs的增殖水平；RT-PCR检测OPG mRNA的表达。结果：淫羊藿苷（0、001-0．1μg／mL）对人PDLCs增殖和OPG mRNA表达具有促进作用（P〈0．01），0．01μg／mL浓度作用最明显。结论：淫羊藿苷能够促进人PDLCs增殖和OPG mRNA表达。