Human periodontal ligament fibroblasts are the optimal cell source for induced pluripotent stem cells

Among the various kinds of fibroblasts existing in the human body, the periodontal ligament (PDL) fibroblasts have been suggested as multipotent cells. Periodontal ligament fibroblasts are characterized by rapid turnover, a high remodeling capacity and remarkable capacity for renewal and repair. They also differentiate into osteoblasts and cementoblasts. We established iPS cells from human PDL fibroblasts by introducing the ES cell markers Oct3/4, Sox2, Nanog, Klf4 and Lin28 by retrovirus transduction, even without the oncogene c-Myc. The iPS cells established in this study expressed the ES cell markers and formed teratomas in SCID mice. The c-Myc expression level in the PDL fibroblasts was higher than that in the iPS cells by quantitative RT-PCR. Therefore, we have concluded that PDL fibroblasts could be an optimal cell source for iPS cells.     

Desmosomal proteins in cultured and intact human periodontal ligament fibroblasts

This study examined the kinds of desmosomal proteins in the human periodontal ligament fibroblasts (PDLFs). The PDLFs obtained from young and older patients were cultured and the amounts of desmosomal proteins were measured by ELISA with antibodies to desmoplakins, desmogleins, and desmocollins. Cultured cells and tissue sections of the human periodontal ligament were immunostained with the same antibodies. Expression of desmosomal proteins in the PDLFs was clearly demonstrated both by ELISA and the immunohistochemical studies, suggesting the existence of desmosome-like junctions in the PDLFs. The junctions are considered to protect gap junctions in the PDLFs against cell transformation caused by cell contraction, which may relate to tooth eruption and repair of periodontal tissue, and/or strong occlusal forces. Statistically significant differences (P < 0.0001) in the expression of desmoplakins and desmogleins between younger and older patients were observed in this study.     

Low-intensity pulsed ultrasound promotes bone morphogenic protein 9-induced osteogenesis and suppresses inhibitory effects of inflammatory cytokines on cellular responses via Rho-associated kinase 1 in human periodontal ligament fibroblasts

Periodontal ligament fibroblasts (PDLFs) have osteogenic capacity, producing bone matrix proteins. Application of bone morphogenic proteins (BMPs) to PDLFs is a promising approach for periodontal regeneration. However, in chronic bone metabolic disorders, such as periodontitis, proper control of accompanying inflammation is essential for optimizing the effects of BMPs on PDLFs. We have previously shown that low-intensity pulsed ultrasound (LIPUS), a medical technology that induces mechanical stress using sound waves, significantly promotes osteogenesis in mesenchymal stem cells. Here, we demonstrate that LIPUS promotes the BMP9-induced osteogenic differentiation of PDLFs. In contrast, BMP2-induced osteogenic differentiation was not altered by LIPUS, probably due to the LIPUS-induced secretion of Noggin, a BMP2 antagonist, from PDLFs. To examine if LIPUS affects inflammatory responses of PDLFs to lipopolysaccharide (LPS) derived from Porphyromonas gingivalis (LPS-PG), we also simultaneously treated PDLFs with LIPUS and LPS-PG. Treatment with LIPUS significantly inhibited the phosphorylation of ERKs, TANK-binding kinase 1, and interferon regulatory factor 3 in LPS-PG-stimulated PDLFs, in addition to inhibiting the degradation of IκB. Furthermore, LIPUS treatment reduced messenger RNA (mRNA) expression of interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, IL-8, C-C motif chemokine ligand 2, C-X-C motif chemokine ligand 1 (CXCL1), CXCL10 and receptor activator of nuclear factor kappa-B ligand, and also diminished IL-1ß and tumor necrosis factor a (TNFa)-induced inflammatory reactions. Phosphorylation of Rho-associated kinase 1 (ROCK1) was induced by LIPUS, while ROCK1-specific inhibitor prevented the promotive effects of LIPUS on p38 phosphorylation, mRNA expression of CXCL1 and Noggin, and osteogenesis. The suppressive effects of LIPUS on LPS-PG-stimulated inflammatory reactions were also prevented by ROCK1 inhibition. Moreover, LIPUS treatment blocked inhibitory effects of LPS-PG and IL-1ß on osteogenesis. These results indicate that LIPUS suppresses inflammatory effects of LPS-PG, IL-1ß, and TNFa and also promotes BMP9-induced osteogenesis through ROCK1 in PDLFs.     

PERIODONTAL LIGAMENT CELL KINETICS FOLLOWING ORTHODONTIC TOOTH MOVEMENT

The population of periodontal ligament (PDL) fibroblasts examined in this study may include osteogenic progenitor cells. PDL fibroblast and osteoblast kinetics in the periodontal ligament of the rat were measured following orthodontic stimulation of bone formation. Both single and multiple injections of tritiated thymidine (³H-TdR) were used. In single injection experiments, the peak percentage of PDL fibroblasts labeled with ³H-TdR is 15% at 22 hr post-stimulation. In multiple injection experiments, the total percentage of fibroblasts in the PDL which respond by synthesizing DNA is 50%. ³H-TdR-Iabeled osteoblasts appear at the same rate as, but with a time delay after, the labeled fibroblasts. Following stimulation, the most likely source of osteoblasts at the bone-forming site is not only fibroblasts which make DNA, divide, then differentiate, but also fibroblasts which either are differentiated to osteoblasts without DNA synthesis and cell division, or are released from G₂ block by the orthodontic stimulation.     

Auto-transplanted mesenchymal stromal cell fate in periodontal tissue of beagle dogs

Background aimsMesenchymal stromal cells (MSC) possess multilineage differentiation potential and characteristics of self-renewal. It has been reported that MSC can acquire characteristics of cells in the periodontal ligament (PDL) in vitro. Moreover, the transplantation of MSC has been shown to be a promising strategy for treating periodontal defects. However, little is known about the fate of MSC in periodontal tissue in vivo. The aim of this study was to trace the paths of MSC after transplantation into periodontal tissues in vivo.MethodsMSC labeled with bromodeoxyuridine (BrdU) were transplanted into periodontal defects of beagle dogs. Six weeks after surgery, the animals were killed and decalcified specimens were prepared. Migration and differentiation of MSC were detected by single/double immunohistochemistry and a combination of immunohistochemistry and in situ hybridization.ResultsBrdU-labeled MSC were observed distributing into periodontal tissue that included alveolar bone, PDL, cementum and blood vessels and expressing surface markers typical of osteoblasts and fibroblasts.ConclusionsCumulatively, our data suggest that MSC migrate throughout periodontal tissue and differentiate into osteoblasts and fibroblasts after transplantation into periodontal defects at 6 weeks in vivo, and have the potential to regenerate periodontal tissue.     

Migration of periodontal ligament fibroblasts on nanometric topographical patterns: influence of filopodia and focal adhesions on contact guidance

Considered to be the "holy grail" of dentistry, regeneration of the periodontal ligament in humans remains a major clinical problem. Removal of bacterial biofilms is commonly achieved using EDTA gels or lasers. One side effect of these treatment regimens is the etching of nanotopographies on the surface of the tooth. However, the response of periodontal ligament fibroblasts to such features has received very little attention. Using laser interference lithography, we fabricated precisely defined topographies with continuous or discontinuous nanogrooves to assess the adhesion, spreading and migration of PDL fibroblasts. PDL fibroblasts adhered to and spread on all tested surfaces, with initial spreading and focal adhesion formation slower on discontinuous nanogrooves. Cells had a significantly smaller planar area on both continuous and discontinuous nanogrooves in comparison with cells on non-patterned controls. At 24 h post seeding, cells on both types of nanogrooves were highly elongated parallel to the groove long axis. Time-lapse video microscopy revealed that PDL fibroblast movement was guided on both types of grooves, but migration velocity was not significantly different from cells cultured on non-patterned controls. Analysis of filopodia formation using time-lapse video microscopy and labeling of vinculin and F-actin revealed that on nanogrooves, filopodia were highly aligned at both ends of the cell, but with increasing time filopodia and membrane protrusions developed at the side of the cell perpendicular to the cell long axis. We conclude that periodontal ligament fibroblasts are sensitive to nanotopographical depths of 85-100 μm, which could be utilized in regeneration of the periodontal ligament.     

周期性张应力对牙周膜成纤维细胞凋亡的影响

目的:在体外条件下,探讨周期张应力作用对人牙周膜成纤维细胞凋亡的影响及PI3k/Akt信号通路在细胞凋亡中的作用。方法:应用多通道细胞牵张应力加载系统,以HPDLFs(人牙周膜成纤维细胞)为对象构建细胞体外培养-力学刺激模型,对照组为0h,0h+LY294002,加力组1 h,6 h,12 h,12 h+LY294002,24 h,力值定为15%,频率为1/6HZ,即10循环/分钟。采用Hoechst33258染色检测细胞形态和凋亡情况,应用RT-PCR技术检测Bcl-2、Bax的表达情况。结果:Hoechst 33258细胞染色结果显示,对照组的细胞核为弥散均匀的圆形或椭圆形荧光,实验组的细胞核或细胞质内出现可见致密浓染的颗粒、新月体或环状荧,RT-PCR结果显示Bcl-2与Bax基因表达均呈现时间依赖性。12 h HPDLFs的细胞凋亡数达最高峰值(P<0.01),24 h细胞凋亡峰值开始下降,但仍高于未加力组(P<0.05)。与对照组相比加入LY294002后,Bcl-2/Bax比值较加载相同时间的加力组小(P<0.05)。结论:一定的时间范围内,周期性张应力能促进HPDLFs凋亡;随着时间的延长(24h),细胞凋亡受到抑制;PI3K/Akt信号传导通路可能参与在周期性张应力介导的HPDLFs的凋亡。     

Mechanical stress regulates osteogenic differentiation and RANKL/OPG ratio in periodontal ligament stem cells by the Wnt/β-catenin pathway

BackgroundThe balance between osteoblastic and osteoclastic activity is critical in orthodontic tooth movement (OTM). Mesenchymal stem cells (MSCs) play an important role in maintaining bone homeostasis, and periodontal ligament stem cells (PDLSCs) are tissue-specific MSCs in the periodontal ligament. However, whether PDLSCs are required for periodontal tissue remodeling during OTM is not fully understood.MethodsHere, we used PDGFRα and Nestin to trace PDLSCs during OTM in rats. We treat human PDLSCs with 100 kpa static pressure for 1 h or 12 h in vitro, and examined the phenotypic changes and expression of RANKL and OPG in these cells.ResultsIn vivo, we found that positive signals of PDGFRα and Nestin in the PDL gradually increased and then decreased on the pressure side to which pressure was applied. In vitro, the osteogenic differentiation of PDLSCs was significantly increased after force treatment for 1 h relative to 12 h. In contrast, the expression ratio of RANKL/OPG was reduced at 1 h and significantly increased at 12 h. Furthermore, we found that the Wnt/β-catenin pathway was dynamically activated in the PDL and in PDLSCs after mechanical stimulation. Importantly, the canonical Wnt pathway inhibitor DKK1 blocked the osteogenesis effect and rescued the ratio of RANKL/OPG in PDLSCs under force treatment for 1 h.ConclusionsOur findings reveal that PDLSCs participate in OTM and that the Wnt/β-catenin pathway maintains bone homeostasis during tooth movement by regulating the balance between osteoblastic and osteoclastic activity.General significanceWe describe a novel potential mechanism related to tooth movement.     

Porphyromonas gingivalis GroEL Induces Osteoclastogenesis of Periodontal Ligament Cells and Enhances Alveolar Bone Resorption in Rats

Porphyromonas gingivalis is a major periodontal pathogen that contains a variety of virulence factors. The antibody titer to P. gingivalis GroEL, a homologue of HSP60, is significantly higher in periodontitis patients than in healthy control subjects, suggesting that P. gingivalis GroEL is a potential stimulator of periodontal disease. However, the specific role of GroEL in periodontal disease remains unclear. Here, we investigated the effect of P. gingivalis GroEL on human periodontal ligament (PDL) cells in vitro, as well as its effect on alveolar bone resorption in rats in vivo. First, we found that stimulation of PDL cells with recombinant GroEL increased the secretion of the bone resorption-associated cytokines interleukin (IL)-6 and IL-8, potentially via NF-κB activation. Furthermore, GroEL could effectively stimulate PDL cell migration, possibly through activation of integrin α1 and α2 mRNA expression as well as cytoskeletal reorganization. Additionally, GroEL may be involved in osteoclastogenesis via receptor activator of nuclear factor κ-B ligand (RANKL) activation and alkaline phosphatase (ALP) mRNA inhibition in PDL cells. Finally, we inoculated GroEL into rat gingiva, and the results of microcomputed tomography (micro-CT) and histomorphometric assays indicated that the administration of GroEL significantly increased inflammation and bone loss. In conclusion, P. gingivalis GroEL may act as a potent virulence factor, contributing to osteoclastogenesis of PDL cells and resulting in periodontal disease with alveolar bone resorption.     

Expression of UNCL during development of periodontal tissue and response of periodontal ligament fibroblasts to mechanical stress in vivo and in vitro

Mutations in two genes, uncoordinated (unc) and uncoordinated-like (uncl), lead to a failure of mechanotransduction in Drosophila. UNCL, the human homolog of unc and uncl, is preferentially expressed in periodontal ligament (PDL) fibroblasts compared with gingival fibroblasts. However, the precise role of UNCL in the PDL remains unclear. The aim of the present study has been to examine whether mechanical stimuli modulate the expression of UNCL in the human PDL in vivo and in vitro and to examine the roles of UNCL in the development, regeneration, and repair of the PDL. We have investigated the expression pattern of UNCL during the development of periodontal tissue and the response of PDL fibroblasts to mechanical stress in vivo and in vitro. The expression of UNCL mRNA and protein increases with PDL fibroblast differentiation from the confluent to multilayer stage but slightly decreases on mineralized nodule formation. UNCL has also been localized in ameloblasts and adjacent cells, differentiating cementoblasts, and osteoblasts of the developing tooth. Strong distinct UNCL expression has further been observed in the differentiating cementoblasts of the tooth periodontium at the site of tension after orthodontic tooth movement. Application of cyclic mechanical stress on PDL fibroblasts increases the expression of UNCL mRNA. These results indicate that UNCL plays important roles in the development, differentiation, and maintenance of periodontal tissues and also suggest a potential role of UNCL in the mechanotransduction of PDL fibroblasts.This work was supported by a grant from the Korea Health 21R&D Project, Ministry of Health & Welfare, Republic of Korea (03-PJ1-PG1-CH08-0001).     

Tenomodulin Expression in the Periodontal Ligament Enhances Cellular Adhesion

Tenomodulin (Tnmd) is a type II transmembrane protein characteristically expressed in dense connective tissues such as tendons and ligaments. Its expression in the periodontal ligament (PDL) has also been demonstrated, though the timing and function remain unclear. We investigated the expression of Tnmd during murine tooth eruption and explored its biological functions in vitro. Tnmd expression was related to the time of eruption when occlusal force was transferred to the teeth and surrounding tissues. Tnmd overexpression enhanced cell adhesion in NIH3T3 and human PDL cells. In addition, Tnmd-knockout fibroblasts showed decreased cell adhesion. In the extracellular portions of Tnmd, the BRICHOS domain or CS region was found to be responsible for Tnmd-mediated enhancement of cell adhesion. These results suggest that Tnmd acts on the maturation or maintenance of the PDL by positively regulating cell adhesion via its BRICHOS domain.     

游离脂肪酸对人牙周膜成纤维细胞增殖的影响

目的:探讨游离脂肪酸(FFAs)对人牙周膜成纤维细胞增殖的影响,研究游离脂肪酸在代谢综合征患者牙周病发病机制中的作用。方法:选用在牙周组织修复中起主要作用的人牙周膜成纤维细胞进行体外培养,对照组加入不含胎牛血清的DMEM,实验组分别加入不同浓度的游离脂肪酸进行刺激,在刺激24h-72h后,采用四甲基偶氮唑蓝比色(MTT)法检测人牙周膜成纤维细胞的增殖情况。结果:与对照组相比,游离脂肪酸可以抑制人牙周膜成纤维细胞的生长增殖(P<0.01),并且这种抑制作用具有浓度和时间依赖性,以培养72h后抑制作用最为明显(P<0.01)。结论:游离脂肪酸可以抑制牙周膜成纤维细胞的增殖,降低代谢综合征患者牙周组织的的修复能力,从而导致或加重牙周病的发生或发展。     

<Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis>-derived lipopolysaccharide-mediated activation of MAPK signaling regulates inflammatory response and differentiation in human periodontal ligament fibroblasts

Porphyromonas gingivalis (P.g.), which is a potential pathogen for periodontal diseases, contains lipopolysaccharide (LPS), and this endotoxin stimulates a variety of cellular responses. At present, P.g.-derived LPS-induced cellular responses in human periodontal ligament fibroblasts (PDLFs) are not well characterized. Here, we demonstrate that P.g-derived LPS regulates inflammatory responses, apoptosis and differentiation in PDLFs. Interleukin-6 (IL-6) and -8 (IL-8) were effectively upregulated by treatment of P.g.-derived LPS, and we confirmed apoptosis markers including elevated cytochrome c levels, active caspase-3 and morphological change in the presence of P.g.-derived LPS. Moreover, when PDLFs were cultured with differentiation media, P.g.-derived LPS reduced the expression of differentiation marker genes, as well as reducing alkaline phosphatase (ALP) activity and mineralization. P.g.-derived LPS-mediated these cellular responses were effectively abolished by treatment of mitogen-activated protein kinase (MAPK) inhibitors. Taken together, our results suggest that P.g.-derived LPS regulates several cellular responses via activation of MAPK signaling pathways in PDLFs.     

糖尿病大鼠牙周膜成纤维细胞组织中TNF-α的表达及意义

目的：通过检测TNF-α在糖尿病大鼠牙周组织中的表达情况,探讨高糖环境下肿瘤坏死因子与口腔疾病发生发展的关系,为临床治疗提供理论依据。方法：选取SD大鼠40只,随机分为实验组（30只）和对照组（10只）。实验组采取腹腔注射链脲佐菌素的方法建立糖尿病大鼠模型,观察大鼠牙周组织的变化情况。采用RT-PCR法检测两组大鼠牙周膜成纤维细胞中TNF-α的表达水平,CCK-8法测定大鼠牙周膜成纤维细胞的增殖情况,分析TNF-α对PDLFs增殖的抑制作用。结果：实验组大鼠牙周组织胶原纤维束排列紊乱,有炎症细胞浸润;对照组大鼠牙龈未见红肿或出血。TNF-α在糖尿病大鼠牙周膜成纤维细胞中呈高表达,在健康大鼠牙周组织中不显著,差异具统计学意义（P〈0.05）。TNF-α可抑制PDLFs增殖,并且成浓度依赖性,随着浓度的增加,TNF-α对PDLFs增殖的抑制作用逐渐增强,差异具统计学意义（P〈0.05）。结论：糖尿病可增加牙周疾病的发病几率,糖尿病患者体内的TNF-α对牙周疾病的严重程度起重要作用。     

Isolation and characterization of cultured human periodental ligament fibroblast-specific cDNAs

The molecular mechanisms that control the function of periodontal ligament (PDL) fibroblasts remain unclear. We speculated that the character of differentiating PDL fibroblasts is defined by the altered expansion of specific genes not found in neighboring gingival fibroblasts in the periodontium. To expand this set, subtractive hybridization was applied between cultured human PDL and gingival fibroblasts to identify genes differentially expressed in PDL. Consequently five candidate clones, PDLs (periodontal ligament specific) 5, -17, -22, -25, and -31 were identified and characterized by homology search, Northern analysis, and in situ hybridization. Although the mRNAs of these clones were expressed by bone marrow cells and rarely by gingival fibroblasts, the highest expression was detected in the PDL cells, which were uniformly distributed throughout the whole PDL. Amongst the five candidate clones, we focused on PDLs17, because it is a hypothetical protein whose biological function has not been reported yet in the database. Polyclonal antiserum raised against PDLs17 peptide was made, and stained the PDL fibroblasts, osteoblast-like cells and stromal cells in the bone marrow, but not gingival fibroblasts. The results suggest that clones, PDLs5, -17, -22, -25, and -31 may be used as PDL fibroblast-specific markers, and that PDLs17 could act as an important factor in the differentiation process of PDL fibroblasts.     

Secreted frizzled-related protein 1 (SFRP1) protects fibroblasts from ceramide-induced apoptosis

Secreted frizzled-related proteins (SFRPs) are soluble proteins that have highly restricted tissue distribution. Although not fully understood, a role of SFRP1 in the regulation of apoptosis has been suggested. Our previous study disclosed a much greater level of SFRP1 expression in periodontal ligament fibroblasts (PDLFs), which have been suggested to maintain a reduced level of apoptosis compared with gingival fibroblasts. We have tested the role of SFRP1 in the regulation of fibroblast apoptosis both in vitro and in vivo. Our data showed that SFRP1 was significantly up-regulated in cultured human PDLFs during ceramide-induced apoptosis. In vivo study demonstrated an increased SFRP1 expression in mice periodontal ligament during force-induced apoptosis. While inhibition of endogenous SFRP1 increased the percentage of cell death in cultured human PDLFs, exogenous SFRP1 substantially reduced apoptosis in cultured human gingival fibroblasts, which do not maintain a high level of endogenous SFRP1 expression. The effect of SFRP1 on apoptosis was linked to the regulation of several apoptosis-related genes, including p53, caspase-3, caspase-9, and BCL-2-interacting killer (BIK). Furthermore our results indicated that the addition of exogenous SFRP1 could reduce the level of apoptosis in dermal fibroblasts in vivo, and this effect was also linked to the regulation of similar apoptosis-related genes as observed in in vitro studies. Collectively our results suggest that the constitutive up-regulation of SFRP1 could be an adaptive cell survival mechanism inherent to functionally specialized fibroblasts, and the addition of SFRP1 may contribute to the inhibition of apoptosis in fibroblast-related cells.     

Periodontal ligament cell kinetics following orthodontic tooth movement.

The population of periodontal ligament (PDL) fibroblasts examined in this study may include osteogenic progenitor cells. PDL fibroblast and osteoblast kinetics in the periodontal ligament of the rat were measured following orthodontic stimulation of bone formation. Both single and multiple injections of tritiated thymidine (3H-TdR) were used. In single injection experiments, the peak percentage of PDL fibroblasts labeled with 3H-TdR is 15% at 22 hr post-stimulation. In multiple injection experiments, the total percentage of fibroblasts in the PDL which respond by synthesizing DNA is 50%. 3H-TdR-labeled osteoblasts appear at the same rate as, but with a time delay after, the labeled fibroblasts. Following stimulation, the most likely source of osteoblasts at the bbone forming site is not only fibroblasts which make DNA, divide, then differentiate, but also fibroblasts which either are differentiated to osteoblasts without DNA synthesis and cell division, or are released from G2 block by the orthodontic stimulation.