MiR-874-3p inhibits osteogenic differentiation of human periodontal ligament fibroblasts through regulating Wnt/ß-catenin pathway.

BACKGROUND/PURPOSE: Previous studies have shown that miR-874 is considered to be an important regulatory factor that participated in osteoclast differentiation. The role of miR-874-3p on osteoclast differentiation of human periodontal ligament fibroblast(hPDLF), however, is still unclear. This study was aimed to delve into the related molecular mechanism of miR-874-3p on hPDLF osteoclast differentiation. MATERIALS AND METHODS: The qRT-PCR assays were applied to check miR-874-3p and WNT3A expression levels during the osteoclast differentiation of hPDLF. Alkaline phosphatase (ALP) activity assays and alizarin red staining assays were applied to appraise the degree of hPDLF osteoclast differentiation. Bioinformatics method and dual-luciferase reporter assay were employed together to anticipate and certify the interaction between miR-874-3p and WNT3A. Western blot assay was applied to examine the ß-catenin and WNT3A expression in transfected hPDLF. RESULTS: In this study, the results indicated that the expression level of miR-874-3p was gradually down-regulated while WNT3A was concomitantly increased during osteogenic differentiation of hPDLF. Overexpression or knockdown of miR-874-3p would inhibit or promote WNT3A and ß-catenin protein expression as well as osteogenic differentiation of hPDLF, respectively. Further research indicated that miR-874-3p directly regulated WNT3A expression via coupling with the 3'-UTR of WNT3A. Finally, upregulation of WNT3A expression levels rescues ß-catenin expression levels and osteogenic differentiation of hPDLF inhibited by miR-874-3p was explored. CONCLUSION: MiR-874-3p inhibits osteogenic differentiation of hPDLF through regulating Wnt/ß-catenin pathway.

UPR attenuates the proinflammatory effect of HPDLF on macrophage polarization.

Human periodontal ligament fibroblast (HPDLF) is a major component of the resident cells in the periodontal microenvironment, and plays important roles in periodontitis through multiple mechanisms. Although lipopolysaccharide (LPS) has been shown to cause endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) in HPDLF, the mechanisms governing HPDLF function in periodontitis are unclear. In this study, we tested the ability of unfolded protein response (UPR) to regulate HPDLF in vitro and examined the underlying mechanisms. We found LPS-pretreated HPDLF induced macrophage polarization toward M1 phenotype. UPR activation reduced the inflammatory cytokine production and downregulated the expression of TLR4 in HPDLF. The phosphorylation of NF-κB p65 and I-κB was also inhibited by UPR activation. Our findings demonstrate that the connection of LPS, UPR, and HPDLF may represent a new concrete theory of innate immunity regulation in periodontal diseases, and suggest that targeting of UPR in HPDLF may be developed as a potent therapy for periodontitis.

Effect of ILK on small-molecule metabolism of human periodontal ligament fibroblasts with mechanical stretching.

BACKGROUND: Mechanical stimuli can cause periodontal tissue reconstruction. Studies have found that changes in metabolites can be the terminal effect of integrin-mediated mechanical signaling. As a key kinase in integrin regulation, integrin-linked kinase (ILK) mediates mechanical signal transduction, which may contribute to metabolite changes. Defining the components of small-molecule metabolites can optimize mechanical stimuli and periodontal tissue reconstruction. Our purpose is to detect the effect of ILK-mediated mechanical signaling on intracellular small-molecule metabolites (amino acids and organic acids) in human periodontal ligament fibroblasts (HPDLFs). METHODS: Primary HPDLFs were isolated by enzyme digestion method. Tensile stresses were applied on HPDLFs in vitro using a Flexcell system. ILK gene in HPDLFs was knocked down by RNA interference (RNAi). Twenty common amino acids and seven organic acids in HPDLFs were analyzed by gas chromatography/mass spectrometry technique. RESULTS: Five amino acids (ie, alanine, glutamine, glutamate, glycine, and threonine) and three organic acids (ie, pyruvate, lactate, and citric acid) were found to be changed remarkably after mechanical stretching. In addition, baseline levels of four amino acids (ie, glutamate, glutamine, threonine, and glycine) and two organic acids (ie, lactate and citric acid) were significantly different in ILK knockdown compared with wild-type HPDLFs. CONCLUSION: This study suggests that five amino acids (ie, alanine, glutamine, glutamate, glycine, and threonine) and three organic acids (ie, pyruvate, lactate, and citric acid) may act as cellular mediators for mechanical signals in HPDLFs. Among them, four amino acids (ie, glutamate, glutamine, threonine, and glycine) and two organic acids (ie, lactate and citric acid) may be closely linked to ILK.

The 3-dimensional attachment of human periodontal ligament fibroblasts on periodontally involved root surface following treatment with EDTA: A SEM study in vitro / 대한치주과학회지

This study evaluated the possibility of the 3-dimensional attachment of human periodontal ligament fibroblasts to a periodntally involved root surface after an EDTA treatment in vitro. The human PDL fibroblasts were isolated from the middle third of the root of periodontally healthy teeth extracted for orthodontic reasons. The cells were cultured in a medium containing Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at 37degrees C in humidified air containing 5% CO2. Eight single-rooted teeth were obtained from patients diagnosed with periodotitis. After scaling and root planing, four teeth were etched with 24% ethylenediaminetetracetic acid (EDTA) for two minutes (Experimental group). The other four teeth were not treated with EDTA and were used as the control group. The human PDL fibroblasts were placed in the total root surface and cultured for 4 weeks. The teeth were fixed in 2.5% glutaraldehyde in PBS before preparation for the scanning electron microscopy (SEM) examination. The human PDL fibroblasts showed a healthy morphology on the root surfaces treated with EDTA (Experimental group) and a relatively unhealthy appearance on the treated root surfaces (Control group). This suggests that EDTA favorably affects the 3-dimensional attachment of human PDL fibroblasts cultured on the root surfaces, which may play an important role in periodontal healing and regeneration.

Periodontal tissue engineering by hPDLF seeding on scaffold / 대한치주과학회지

Human periodontal ligament fibroblasts (hPDLF) are very important for curing the periodontal tissue because they can be differentiated into various cells. A tissue engineering approach using a cell-scaffold is essential for comprehending today's periodontal tissue regeneration procedure. This study examined the possibility of using an acellular dermal matrix as a scaffold for human periodontal ligament fibroblast (hPDLF). The hPDLF was isolated from the middle third of the root of periodontally healthy teeth extracted for orthodontic reasons. The cells were cultured in a medium containing Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at 37degrees C in humidified air with 5% CO2. The acellular dermal matrix(ADM) was provided by the US tissue banks(USA). Second passage cells were used in this study. The hPDLF cells were cultured with the acellular dermal matrix for 2 days, and the dermal matrix cultured by the hPDLF was transferred to a new petri dish and used as the experimental group. The control group was cultured without the acellular dermal matrix. The control and experimental cells were cultured for six weeks. The hPDLF cultured on the acellular dermal matrix was observed by Transmission Electron microscopy (TEM). Electron micrography shows that the hPDLF was proliferated on the acellular dermal matrix. This study suggests that the acellular dermal matrix can be used as a scaffold for hPDLF.

Profiling of genes in healthy hGF, aging hGF, healthy hPDLF and inflammatory hPDLF by DNA microarray / 대한치주과학회지

No abstract available.

Cellular study of replicative senescence in human periodontal ligament fibroblast using molecular biology / 대한치주과학회지

Human periodontal ligament fibroblast(hPDLF) is very important to cure periodontal tissue because it can be diverged into various cells. This study examined the expression of MMP-1, TIMP-1, periodontal ligament specific PDLs22, Type I collagen, Fibronectin, TIMP-2, telomerase mRNA in a replicative senescence of hPDLF. The periodontal ligament tissue was obtained from periodontally healthy and non-carious human teeth extracted for orthodontic reasons at the Chosun University Hospital of Dentistry with the donors' informed consent. The hPDLF cells were cultured in a medium containing Dulbecco's modified Eagle medium(DMEM, Gibco BRL, USA) supplemented with 10% fetal bovine serum(FBS, Gibco BRL, USA) at 37C in humidified air with 5% CO2. For the reverse transcription-polymerase chain reaction(RT-PCR) analysis, the total RNA of the 2, 4, 8, 16, 18, and 21 passage cells was extracted using a Trizol Reagent(Invitrogen, USA) in replicative hPDL cells. Two passage cells, i.e. young cells, served as the control, and beta-actin served as the internal control for RT-PCR The results of this study about cell morphology and gene expression according to aging of hPDLF using RT-PCR method are as follows: 1. The size of hPDLF was increased with aging and it was showed that the hPDLF was dying in the final passage. 2. PDLs22 mRNA was expressed in young hPDLF of the two, four, and six passage. 3. TIMP-1 mRNA was expressed in young hPDLF of the two and four passage. 4. There was a tendency that MMP-1 mRNA was weakly expressed over eighteen. 5. Type 1 collagen mRNA was expressed in almost all passages, but it was not expressed in the final passage. 6. Fibronectin mRNA was observed in all passages and it was weakly expressed in the final passage. 7. TIMP-2 and telomerase mRNA were not expressed in this study. Based on above results, it was observed that PDLs22, Type 1 collagen, Fibronectin, MMP-1. and TIMP-1 mRNA in hPDLF were expressed differently with aging. The study using the hPDLF that is collected from healthy patients and periodontitis patients needs in further study.

Cellular study of replicative senescence in human periodontal ligament fibroblast using molecular biology / 대한치주과학회지

Human periodontal ligament fibroblast(hPDLF) is very important to cure periodontal tissue because it can be diverged into various cells. This study examined the expression of MMP-1, TIMP-1, periodontal ligament specific PDLs22, Type I collagen, Fibronectin, TIMP-2, telomerase mRNA in a replicative senescence of hPDLF. The periodontal ligament tissue was obtained from periodontally healthy and non-carious human teeth extracted for orthodontic reasons at the Chosun University Hospital of Dentistry with the donors' informed consent. The hPDLF cells were cultured in a medium containing Dulbecco's modified Eagle medium(DMEM, Gibco BRL, USA) supplemented with 10% fetal bovine serum(FBS, Gibco BRL, USA) at 37C in humidified air with 5% CO2. For the reverse transcription-polymerase chain reaction(RT-PCR) analysis, the total RNA of the 2, 4, 8, 16, 18, and 21 passage cells was extracted using a Trizol Reagent(Invitrogen, USA) in replicative hPDL cells. Two passage cells, i.e. young cells, served as the control, and beta-actin served as the internal control for RT-PCR The results of this study about cell morphology and gene expression according to aging of hPDLF using RT-PCR method are as follows: 1. The size of hPDLF was increased with aging and it was showed that the hPDLF was dying in the final passage. 2. PDLs22 mRNA was expressed in young hPDLF of the two, four, and six passage. 3. TIMP-1 mRNA was expressed in young hPDLF of the two and four passage. 4. There was a tendency that MMP-1 mRNA was weakly expressed over eighteen. 5. Type 1 collagen mRNA was expressed in almost all passages, but it was not expressed in the final passage. 6. Fibronectin mRNA was observed in all passages and it was weakly expressed in the final passage. 7. TIMP-2 and telomerase mRNA were not expressed in this study. Based on above results, it was observed that PDLs22, Type 1 collagen, Fibronectin, MMP-1. and TIMP-1 mRNA in hPDLF were expressed differently with aging. The study using the hPDLF that is collected from healthy patients and periodontitis patients needs in further study.