Periostin对人牙周膜细胞增殖和碱性磷酸酶活性的影响

目的：研究小鼠重组Periostin对牙周膜细胞（PDL细胞）增殖及碱性磷酸酶（ALP）活性的影响。方法：采用MTT比色试验及酶动力学方法。测定PDL细胞的增殖和ALP活性。结果：Periostin仅在浓度为40μg/ml时,可显著促进PLD细胞的增殖（P＜0．01）,其余浓度则无作用,而其浓度在10μg/ml-80μg/ml范围中时,均可显著提高PDL细胞ALP活性水平（P＜0．05）。结论：小鼠重组Periostin对PDL细胞的增殖及ALP活性均有促进作用,但其在这两方面的作用存在着异,对此还需深入研究。     

Periostin对人牙周膜细胞在牙根片上附着和生长的影响

目的：研究小鼠重组Periostin对人牙周膜细胞（PDL细胞）在正常及病变牙根片上附着和增殖的影响。方法：将正常及病变根片分别用Periostin、FN和无血清DMEM处理后,观察PDL细胞在其上24h的附着量和72h增殖量,并在扫描电镜下观察其形态变化,结果：Periostin与阳性对照FN均可有效促进PDL细胞在政党及病变根面上的附着和增殖（P＜0．05－0．01）,二者促附着作用相近（P＞0．05）,但在促增殖方面,Periostin较FN作用明显弱（P＜0．05）。结论：小鼠重组Periostin对PLD细胞在正常及病变根片上的附着和增殖均有一定促进作用。     

小鼠重组Periostin对成骨细胞附着和伸展的影响

目的：观察小鼠重组Periostin对成骨细胞附着和伸展的作用。方法：采用固相结合分析实验和四唑盐比色（MTT）法等细胞生物学技术,观察成骨细胞在Periostin基质上的附着能力和伸展率。结果：成骨细胞在Periostin基质上附着和伸展良好,当浓度在20μg/ml以上时,作用尤其显著（P＜0．01）,与在纤维结合蛋白（Fn）基质上生长相似。结果：小鼠重组Periostin可有效促进成骨细胞的附着和伸展,但其作用的附着和伸展,但其作用的切分子机制还需深入研究。     

Periostin对人牙周膜细胞增殖和碱性磷酸酶活性的影响

目的 :研究小鼠重组Periostin对牙周膜细胞 (PDL细胞 )增殖及碱性磷酸酶 (ALP)活性的影响。方法 :采用MTT比色试验及酶动力学方法 ,测定PDL细胞的增殖和ALP活性。结果 :Periostin仅在浓度为 40 μg/ml时 ,可显著促进PDL细胞的增殖 (P <0 .0 1) ,其余浓度则无作用 ,而其浓度在 10 μg/ml～ 80 μg/ml范围中时 ,均可显著提高PDL细胞ALP活性水平 (P <0 .0 5 )。结论 :小鼠重组Periostin对PDL细胞的增殖及ALP活性均有促进作用 ,但其在这两方面的作用存在差异 ,对此还需深入研究     

小鼠重组OSF-2对人牙周膜细胞附着和伸展的影响

目的：观察小鼠重组牙周膜细胞特异性因子2（OSF-2）对牙周膜细胞附着和伸展的作用。方法：采用固相结合分析实验及四唑盐比色等细胞生物学技术,观察牙周膜细胞在OSF-2基质上的附着能力和伸展率。结果：牙周膜细胞在OSF-2基质上附着和伸展良好,当浓度在20ug／ml以上时,作用尤其显著（P〈0．01）,与在FN基质的生长结果相似。结论：小鼠重组OSF-2可有效促进牙周膜细胞的附着和伸展。     

Periostin抗体的制备和Periostin组织表达特异性的研究

目的:制备抗Periostin抗体,并了解Periostin的组织表达特性。方法:以重组、纯化的小鼠Periostin为抗原,混入Freund完全佐剂充分乳化后,免疫新西兰大白兔;用双向免疫扩散试验检测抗体效价,将获得的多克隆抗血清初步纯化后,用Western blot和免疫组化检测小鼠Periostin的组织表达特异性。结果:Western blot显示Periostin在小鼠颅盖骨及牙周组织总蛋白中有特异性表达,而在肝、肾和脑组织总蛋白中没有表达。免疫组化检测,下颌骨表面骨膜和牙周膜组织中的Periostin染色呈强阳性,而在牙骨质、牙本质、牙槽骨和牙髓组织中则为阴性,并且它不是粘附于细胞表面,而是分布在由成骨细胞和牙周膜细胞分泌于其周围的细胞外基质中,牙槽骨中的骨细胞也不产生Periostin。结论:Periostin这种在有骨形成能力组织中的高表达,而在成熟骨组织中无表达的特点提示了其可能与矿化组织的早期形成有关。     

牙周优势菌内毒素对人牙周膜细胞增殖和碱性磷酸酶活性的影响

目的：研究牙周优势菌牙龈卟啉菌、中间普氏菌、具核梭杆菌的内毒素对人牙周膜细胞（PDL细胞）增殖和碱性磷酸酶活性（ALP）的影响。方法：采用MTT比色试验及酶动力学方法，测定PDL细胞的增殖和ALP活性。结果：内毒素在10μg/mL、100μg/mL高浓度时，可显著抑制PDL细胞增殖，而在0.01μg/mL、0.1μg/mL低浓度时，则促进PDL细胞增殖；在10μg/mL、100μg/mL可呈浓度依赖性方式抑制PDL细胞ALP活性。结论：内毒素对牙周膜细胞的抑制作用主要和其浓度有关，不同来源内毒素差异并不显著；内毒素可能通过影响PDL细胞功能而影响牙周组织的代谢和修复过程。     

胰岛素和转化生长因子-β对人牙周膜细胞碱性磷酸酶活性和总蛋白含量的影响

目的：探讨胰岛素和转化生长因子－β（TGF－β）对体外生长的人牙周膜（PDL）细胞生物学特征的影响,方法：通过体外细胞培养技术,酶动力学法和考马斯亮蓝法,检测胰岛素和TGF－β单独或联合应用时对人PDL细胞碱性磷酸酶（ALPase)活性和总蛋白含量的影响。结果：单独应用时,胰岛素浓度1．0－100U／L,TGF－β浓度0．1－100ug/L,明显促进人PDL细胞的ALPase活性和总蛋白含量,最佳浓度分别为10U／L和1ug/L,以二者的最佳浓度联合应用时,作用更为显著（P＜0．01）,结论：胰岛素和TGF－β均能促进人PDL细胞的分化和总蛋白合成功能,二者联合应用有协同效应。β     

釉基质蛋白对大鼠外胚间充质细胞黏附、伸展影响的体外研究

目的：了解体外研究环境中，两种不同形式釉基质蛋白（粗提型EMPs和纯化型EMD）对外胚间充质细胞黏附、伸展行为的影响力。方法：酶消化法培养SD大鼠颌突外胚间充质细胞，采用固相结合分析方法，观察外胚间充质细胞在不同浓度釉基质蛋白（50、100、150及200μg／mL等浓度的EMPs和EMD）包被的培养孔表面黏附、伸展情况，依次命名为ED2、ED2、ED3、ED4及EP1、EP2、EP3、EP4组。结果：①细胞黏附性实验中，仅ED2和ED3组结果高于空白对照组（P〈0．01）。②细胞伸展实验中，EP1、EP2和EP3组结果高于空白对照组（P〈0.01）。结论：EMD对外胚间充质细胞伸展无明显促进作用，但可显著促进其黏附贴壁；EMPs对外胚间充质细胞黏附无影响，但可促进其伸展。     

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。     

Biomimetic approach on human periodontal ligament cells using synthetic oligopeptides

BACKGROUND: Periodontal ligament (PDL) cells, connecting root cementum with alveolar bone, are important for periodontal wound healing. In order to obtain a predictable periodontal regeneration, selective adhesion and proliferation of PDL cells are essential. The purpose of this study was to investigate the effects of synthetic peptides mimicking cell-binding domain of fibronectin (FN) on human PDL cells. METHODS: Two types of oligopeptides, Gly3-Pro-His-Ser-Arg-Asn-Gly6-Arg-Gly-Asp-Gly (G3PHSRNG6RGDG) and Gly3-His-Pro-Asn-Arg-Ser-Gly6-Arg-Gly-Asp-Gly (G3HPNRSG6RGDG), were constructed using a solid-phase peptide synthesizer. Fibronectin type III ninth to tenth domain (FN III 9-10) and Arg-Gly-Asp-Ser (RGDS) were prepared for comparison with the effects of synthetic peptides. These peptides were coated onto 96-well cell culture plates with 0.001 approximately 100 microM concentrations. Cultured human PDL cells were then applied to the peptide-coated wells at a density of 1 x 10(4)/well. After 1 hour incubation at 37 degrees C, adhered cells were fixed, stained, and examined by phase contrast microscopy for cell spreading assay. Attached PDL cells were solubilized with 2% sodium dodecyl sulfate (SDS) for the cell attachment assay by measuring absorbance at 595 nm in microplate reader. Western blot analysis was performed to determine extracellular signal-regulated kinase (ERK1/2) activity. RESULTS: Cell attachment and spreading assays revealed that G3PHSRNG6RGDG (> or = 10 microM) possesses similar adhesive behavior to FN III 9-10. G3PHSRNG6RGDG showed a comparable ERK1/2 activity when compared to FN III 9-10. CONCLUSIONS: G3PHSRNG6RGDG enhanced an attachment and spreading of human PDL cells thereby increasing ERK1/2 activity. Taken together, it is anticipated that this peptide might be a potential tool for arranging a biologically attractive environment for PDL cells, which would enhance periodontal regeneration efficacy.     

Effects of enamel matrix derivative and transforming growth factor-beta1 on human periodontal ligament fibroblasts

AIM: The objective of this study was to evaluate the effects of enamel matrix derivative (EMD), transforming growth factor-beta1 (TGF-beta1), and a combination of both factors (EMD+TGF-beta1) on periodontal ligament (PDL) fibroblasts. MATERIAL AND METHODS: Human PDL fibroblasts were obtained from three adult patients with a clinically healthy periodontium, using the explant technique. The effects of EMD, TGF-beta1, or a combination of both were analysed on PDL cell proliferation, adhesion, wound healing, and total protein synthesis, and on alkaline phosphatase (ALP) activity and bone-like nodule formation. RESULTS: Treatment with EMD for 4, 7, and 10 days increased cell proliferation significantly compared with the negative control (p<0.05). At day 10, EMD and EMD+TGF-beta1 showed a higher cell proliferation compared with TGF-beta1 (p<0.01). Cell adhesion was significantly up-regulated by TGF-beta1 compared with EMD and EMD+TGF-beta1 (p<0.01). EMD enhanced in vitro wound healing of PDL cells compared with the other treatments. Total protein synthesis was significantly increased in PDL cells cultured with EMD compared with PDL cells treated with TGF-beta1 or EMD+TGF-beta1 (p<0.05). EMD induced ALP activity in PDL fibroblasts, which was associated with an increase of bone-like nodules. CONCLUSION: These findings support the hypothesis that EMD and TGF-beta1 may play an important role in periodontal regeneration. EMD induced PDL fibroblast proliferation and migration, total protein synthesis, ALP activity, and mineralization, while TGF-beta1 increased cellular adhesion. However, the combination of both factors did not positively alter PDL fibroblast behaviour.     

Tumor Necrosis Factor‐α and Porphyromonas gingivalis Lipopolysaccharides Decrease Periostin in Human Periodontal Ligament Fibroblasts

Background: Periostin is a matricellular protein essential for tissue integrity and maturation and is believed to have a key function as a modulator of periodontal ligament (PDL) homeostasis. The aim of this study is to evaluate whether periodontal disease‐associated pathogen‐related virulence factors (endotoxins/lipopolysaccharides [LPS]) and proinflammatory cytokines alter the expression of periostin in PDL cells. Methods: Human PDL cultures were exposed to inflammatory mediators (tumor necrosis factor‐α [TNF‐α]), bacterial virulence factors (Porphyromonas gingivalis LPS) or a combination in a biomechanically challenged environment. Culture conditions were applied for 24 hours, 4 days, and 7 days. Periostin and TGF‐β inducible gene clone H3 (βIGH3) mRNA expression from cell lysates were analyzed. Periostin and βIGH3 proteins were also detected and semiquantified in both cell lysates and cell culture supernatants by Western blot. In addition, periostin localization by immunofluorescence was performed. Analysis of variance and Fisher tests were used to define the statistical differences among groups (P <0.05). Results: In a mechanically challenged environment, periostin protein was more efficiently incorporated into the matrix compared to the non‐loaded controls (higher levels of periostin in the supernatant in the non‐loaded group). Interestingly, chronic exposure to proinflammatory cytokines and/or microbial virulence factors significantly decreased periostin protein levels in the loaded cultures. There was greater variability on βIGH3 levels, and no particular pattern was clearly evident. Conclusions: Inflammatory mediators (TNF‐α) and bacterial virulence factors (P. gingivalis LPS) decrease periostin expression in human PDL fibroblasts. These results support a potential mechanism by which periostin alterations could act as a contributing factor during periodontal disease progression.     

Chemotactic response of periodontal ligament cells decreases with donor age: association with reduced expression of c-fos

OBJECTIVE: To understand the effects of aging on cellular motility of human periodontal ligament (PDL) cells, and to determine the possible link between cell proliferation and migration in relation to cellular aging. MATERIALS AND METHODS: The chemotactic response of PDL cells from three juvenile and four older donors were compared. Then, migrated or unmigrated cells were examined for their cell cycle by morphological and immunocytochemical procedures. Finally, migrated or unmigrated cells were examined for the expression of c-fos and c-myc by in situhybridization. RESULTS: PDL cells from older donors showed lower chemotaxis compared with the cells from juvenile donors (P < 0.05). Cells undergoing migration were found not to be in the S- or M-phase of the cell cycle. However, all migrated cells were found to express c-fos, while many of the cells which did not migrate were found not to express c-fos. CONCLUSIONS: Cellular motility of PDL cells decreases with donor age as well as cell proliferation. Since the cells reaching senescence fail to express c-fos, the mechanisms linked to cellular senescence may be a possible underlying mechanism for low migration seen in the older cells.     

Inhibitory effects of tumor necrosis factor-alpha on migration of human periodontal ligament cells

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is associated with chronic gingival inflammation and is suspected to influence periodontal destruction. However, the exact roles of TNF-alpha in wound healing and periodontal tissue regeneration are largely unknown. In the present study, we examined the effects of TNF-alpha on migration and proliferation of human periodontal ligament (PDL) cells. METHODS: PDL cells were cultured in the presence of TNF-alpha to determine its effects on cellular migration and proliferation. The protein expression profiles of alpha5 and beta1 integrin subunits and their related molecules, paxillin and focal adhesion kinases (FAK), were investigated. Gene expression of fibronectin also was assayed. Further, the activation of Rho-family small guanosine triphosphate (GTP)-binding protein (RhoA) was evaluated using a GTP-loading pull-down assay, and focal adhesion formation by PDL cells after transfection with the expression vector of paxillin-fused green fluorescent protein (GFP) also was observed with confocal microscopy. RESULTS: Cellular migration was impaired by TNF-alpha and recovered following the addition of anti-TNF-alpha antibodies. In contrast, PDL cell proliferation was not affected by TNF-alpha. TNF-alpha upregulated the expression of the alpha5 and beta1 integrin subunits, whereas fibronectin was not overexpressed. Phosphorylation of paxillin and FAK by PDL cells was induced, and RhoA activation also was induced. Confocal microscopic analysis revealed that TNF-alpha induced focal adhesion and stress fiber formation in all parts of the cells. CONCLUSION: Our results suggested that TNF-alpha impairs cellular migration by enhancing cellular adhesive ability following significant focal adhesion and stress fiber formation.     

Greater Sensitivity of Oral Fibroblasts to Smoked Versus Smokeless Tobacco

Background: Smokers have an increased incidence and severity of periodontal disease. Although cigarette smoke contains >4,000 chemical components that could affect periodontal tissues, less is understood about the effect of smokeless tobacco. Therefore, this study compares the effects of cigarette smoke extract (CSE) and smokeless tobacco extract (STE) on cell survival and motility of periodontal ligament (PDL) and gingival fibroblasts in vitro. Methods: PDL and gingival fibroblasts were exposed to various concentrations of CSE, STE, or nicotine alone. Viable cells were labeled with calcein acetoxymethyl, visualized using fluorescent microscopy, and quantified using a fluorescence multi‐well plate reader. In vitro wounding and collagen gel contraction assays were used to assess cell motility. Results: Both gingival and PDL fibroblasts displayed reduced cell viability with increasing concentrations of CSE and STE. Based on relative nicotine content, CSE was significantly more cytotoxic than STE. PDL fibroblasts were also more sensitive to both CSE and STE compared with gingival fibroblasts. Finally, sublethal doses of CSE reduced cell motility and gel contraction, whereas STE had less effect. Nicotine alone ≤0.5 mM had little to no effect in any of these assays. Conclusions: Many of the underlying effects of tobacco products on periodontal tissues may be due to direct inhibition of normal fibroblast function. CSE is found to be more deleterious to the function of both PDL and gingival fibroblasts than STE. PDL fibroblasts appear to be more sensitive to CSE and STE than gingival fibroblasts. Therefore, cigarette smoke may have more profound effects than smokeless tobacco.     

Proliferation and adhesion of periodontal ligament cells on synthetic biominerals

OBJECTIVE: Hydroxiapatite (HA) has been suggested as a useful biomaterial to support the regeneration of tissues. In this study, we investigated the adhesion of periodontal ligament (PDL) cells on octacalcium phosphate (OCP) and its hydrolyzed apatitic product (HL), which are known precursors of HA. METHODS: Rat PDL cells were cultured on OCP or HL-coated dishes. Cell proliferation and adhesion and mRNA expression of collagen I, fibronectin integrin subunits were examined. Cell adhesion inhibition assays were carried out by GRGDSPK (Gly-Arg-Gly-Asp-Ser-Pro-Lys). RESULTS: In early culture period, the cell number of PDL cells was lower on OCP and HL than that on control without any coating. However, the cell number on OCP or HL caught up with control later period. mRNA expression level of collagen I and fibronectin on OCP and HL were similar among OCP HL and control, although they differed early in the culture period. Integrin subunits were expressed on both OCP and HL as well as on control. Cell adhesion was inhibited by RGD inhibitor peptide. CONCLUSION: Our findings indicated that rat PDL cells produce collagen I and fibronectin on OCP and HL, and then show increased cell numbers depending on adhesion to the matrices through integrins.