• Journal of Periodontal and Implant Science
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• v.40 no.6
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• pp.265-270
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• 2010

Purpose: The aim of this study was to investigate the immunomodulatory effects of canine periodontal ligament stem cells on allogenic and xenogenic immune cells in vitro. Methods: Mixed cell cultures consisting of canine stem cells (periodontal ligament stem cells and bone marrow stem cells) and allogenic canine/xenogenic human peripheral blood mononuclear cells (PBMCs) were established following the addition of phytohemagglutinin. The proliferation of PBMCs was evaluated using the MTS assay. The cell division of PBMCs was analyzed using the CFSE assay. The apoptosis of PBMCs was assessed using the trypan blue uptake method. Results: Periodontal ligament stem cells and bone marrow stem cells inhibited the proliferation of allogenic and xenogenic PBMCs. Both periodontal ligament stem cells and bone marrow stem cells suppressed the cell division of PBMCs despite the existence of a mitogen. No significant differences in the percentages of apoptotic PBMCs were found among the groups. Conclusions: Canine periodontal ligament stem cells have an immunomodulatory effect on allogenic and xenogenic PBMCs. This effect is not a product of apoptosis of PBMCs but is caused by the inhibition of cell division of PBMCs.

• Journal of Periodontal and Implant Science
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• v.47 no.6
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• pp.402-415
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• 2017

Purpose: The aim of this study was to determine the effects of patterned human periodontal ligament stem cell (hPDLSC) sheets fabricated using a thermoresponsive substratum. Methods: In this study, we fabricated patterned hPDLSC sheets using nanotopographical cues to modulate the alignment of the cell sheet. Results: The hPDLSCs showed rapid monolayer formation on various surface pattern widths. Compared to cell sheets grown on flat surfaces, there were no significant differences in cell attachment and growth on the nanopatterned substratum. However, the patterned hPDLSC sheets showed higher periodontal ligamentogenesis-related gene expression in early stages than the unpatterned cell sheets. Conclusions: This experiment confirmed that patterned cell sheets provide flexibility in designing hPDLSC sheets, and that these stem cell sheets may be candidates for application in periodontal regenerative therapy.

• Journal of Periodontal and Implant Science
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• v.25 no.3
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• pp.539-556
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• 1995

Periodontal therapeutic modalities should be re-establishing and regenerating the periodontal tissue previously lost to the disease. To achieve periodontal regeneration, periodontal ligament cells must selective migrate to the deneded root surface, attached and proliferated it. Local pH concentration is one of the most factors that periodontal regeneration. The aims of this study were to examine on biological effects of pH to the human periodontal ligament cells in vitro, especially on the cell morphology, attachment, activity, vitality and viability. Human periodontal ligament cells were cultured from extracted tooth for non-periodontal reason. Immediately after extraction, any soft tissue adhering to the cervical parts of the roots was carefully removed with a sterile curette. To produce different pH levels in the media, Eagle's MEM was adjusted from pH 6.6 to 8.2 in 0.2 intervals with 1 M NaOH and 1 N HCl. After cultivation, Then, Periodontal ligament cells were cultured at pH ranging from 6.6-8.2. attachment assay was done at 1, 2 day incubation and activity assay was done at 1, 2, 3 day incubation. The experiments were evaluated by scaning electron microscopic techniques (HITACHIX-650 Scaning Electron Microanalyzer, Tokyo, Japan), MTT assay, and the cultured periodontal ligament cells were fixed in neutral formalin for 24 hours and immunohistochemically processed by PCNA for proliferating ability. The surviving cells in the medium showed slightly increased volume and widening intercellular distances at low concentration of pH than control group (pH 7.4), and apparently shrinkage at high concentration of pH than control group (pH 7.4). The results of the statistical analysis from the experiment on attachment, vitality and viability were as follows. Attachment of periodontal ligament cells at 1st and 2nd day, similar attachment rate of low concentration pH compared with control value (pH 7.4). But above pH 8.0, attachment rate were statistically significant decrease from control value(P<0.05). Periodontal ligament cell's activities were maximum at pH 7.6 by MTT assay. Similar with control value at low concentration of pH. But, the activities were statistically significant decrease at high concentraration of pH(P<0.05). Cellular proliferating rate (PCNA index) were statistically significant decrease from control value at low and high concentration of pH(p<0.05). This results suggested that hjgh concentration pH, in other words, alkali pH was cytotoxic effects on human periodontal ligament cells in vitro.

• Journal of Periodontal and Implant Science
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• v.36 no.2
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• pp.335-344
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• 2006

Osteoblast or bone marrow stromal cell-derived RANKL is the major effector molecule essential for osteoclastogenesis. Previous studies have shown that tetracyclines have beneficial therapeutic effects in the prevention and treatment of inflammatory bone disease including periodontal disease. Periodontal ligament cells are thought not only to play an important role in the progression of periodontal disease, but to play an important role in alveolar bone remodeling. Previous studies indicated that receptor activation of nuclear factor $\kappa\;B$ ligand (RANKL) and osteoprotegerin (OPG) are expressed in periodontal ligament cells by pro-inflammatory cytokine, such as $IL-1{\beta}$ and $TNF-{\alpha}$. This study was designed to investigate the inhibitory effect of doxycycline on RANKL and OPG mRNA in rat periodontal ligament cells induced by $IL-1{\beta}$ (1 ng/ml). The results are as follows; 1. MTT assay showed that doxycycline at the concentration of $1-50\;{\mu}g/m{\ell}$ didn't result in statistically significant cell death at day 1 and 3. 2. RANKL mRNA expression was increased to 2.6 folds by $IL-1{\beta}$. When cells were treated with doxycycline ($50{\mu}g/m{\ell}$), $IL-1{\beta}$ -induced mRANKL expression was reduced by 33%. In contrast to RANKL, OPG mRNA expression was not inhibited by pre-treatment with doxycycline. These results suggest that doxycycline decrease the expression of mRANKL resulting in regulation of osteoclastogenesisp in rat periodontal ligament cells.

• Journal of Periodontal and Implant Science
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• v.41 no.2
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• pp.73-78
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• 2011

Purpose: For periodontal tissue engineering, it is a primary requisite and a challenge to select the optimum types of cells, properties of scaffold, and growth factor combination to reconstruct a specific tissue in its natural form and with the appropriate function. Owing to fundamental disadvantages associated with using a two-dimensional substrate, several methods of seeding cells into three-dimensional scaffolds have been reported and the authors have asserted its usefulness and effectiveness. In this study, we explore the cell attachment of periodontal ligament fibroblasts on nanohydroxyapatite (n-HA) scaffold using avidin biotin binding system (ABBS). Methods: Human periodontal ligament fibroblasts were isolated from the health tooth extracted for the purpose of orthodontic procedure. HA nanoparticles were prepared and $Ca(NO_3)_2-_4H_2O$ and $(OC_2H_5)_3P$ were selected as precursors of HA sol. The final scaffold was 8 mm in diameter and 3 mm in height disk with porosity value of 81.55%. $1{\times}10^5$ periodontal ligament fibroblasts were applied to each scaffold. The cells were seeded into scaffolds by static, agitating and ABBS seeding method. Results: The number of periodontal ligament fibroblasts attached was greater for ABBS seeding method than for static or agitating method (P<0.05). No meaningful difference has been observed among seeding methods with scanning electron microscopy images. However, increased strength of cell attachment of ABBS could be deduced from the high affinity between avidin and biotin ($Kd=10^{-15}\;M$). Conclusions: The high-affinity ABBS enhances the ability of periodontal ligament fibroblasts to attach to three-dimensionally constructed n-HA scaffold.

• Journal of Periodontal and Implant Science
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• v.32 no.2
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• pp.389-402
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• 2002

BMP can induce ectopic bone formation when implanted into sites such as rat muscle and can greatly enhance healing of bony defects when applied exogenously. In addition, BMP stimulated osteoblastic differentiation in vitro in various types of cells. The aim of this study was to investigate the effect of recombinant human bone morphogenetic protein(rhBMP-2) on the proliferation and osteoblastic differentiation of human periodontal ligament cells and gingival fibroblasts. The cell number and alkaline phosphatase activity were measured in 3 experimental groups of human periodontal ligament cells and gingival fibroblasts (control group, rhBMP-2 50ng/ml group, and rhBMP-2 100ng/ml group) at 1 and 2 weeks after culture. At the same time, total RNA of cultured cells were extracted and reverse trascription polymerase chain reaction(RT-PCR) was performed to determine the expression of mRNA of bone matrix protein. RhBMP-2 had no effect on the cell proliferation of human periodontal ligament cells and gingival fibroblasts. Alkaline phosphatase activity was elevated significantly by rhBMP-2 in both cells. And periodontal ligament cells showed significantly higher alkaline phosphatase activity than gingival fibroblasts. ${\beta}$-actin, type I collagen, alkaline phosphatase, BMP-2 mRNA were expressed in all of the samples. Osteopontin, osteocalcin mRNA were expressed in all periodontal ligament cell groups, and rhBMP-2 50ng/ml group, rhBMP-2 100ng/ml group of 2 week culture period of gingival fibroblasts. Bone sialoprotein mRNA was only expressed in rhBMP-2 50ng/ml group and rhBMP-2 100ng/ml group of 2-week culture period. These results suggest that rhBMP-2 stimulates osteoblastic differentiation in human periodontal ligament cells and gingival fibroblasts in vitro.

• Journal of Periodontal and Implant Science
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• v.29 no.4
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• pp.821-838
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• 1999

On the basis of the evidences that electrical stimulation could enhance proliferation and differentiation of bone cells and promote healing and regeneration of bone, this study was performed to investigate the effects of electrical stimulation on human periodontal ligament cells and gingival fibroblasts in vitro, which also have important roles in regeneration of periodontium, and to evaluate the potential of clinical application of electrical stimulation. Human periodontal ligament cells and gingival fibroblasts were primarily cultured from the root surface of extracted premolar and the adjacent gingiva without periodontal diseases. In control group, the cells ($5{\times}10^4$ cells/ml)were incubated only in Dulbecco's Modified Eagle's Medium contained with 10% fetal bovine serum. In test groups, electrical stimulation was given at the current intensity of $0.25{\mu]A$(test group 1), $1.0{\mu}A$(test group 2), and $2.5{\mu}A$(test group 3) for 12 hours to the same culture media with the control group. After 12 hour exposure of electrical stimulation, the cells were incubated for 2 and a half days(60 hours), and then each group of cells was analyzed for cell proliferation, protein level, and activity of alkaline phosphatase. The results were as follows ; 1. The Rate of cell proliferation of every test group increased significantly in both periodontal ligament cells and gingival fibroblasts, and in periodontal ligament cells, test group 3 showed significantly increased proliferation compared to the other test groups(p<0.05). 2. In the protein levels, neither periodontal ligament cell nor gingival fibroblast showed statistically significant differences between control and test groups. 3. The activity of alkaline phosphatase in periodontal ligament cells increased significantly in all test groups(p<0.05), but there were no significant differences between 3 test groups. In gingival fibroblasts, the activity of alkaline phosphatase increased significantly only in test group 3(p<0.05). From the above results, it is concluded that electrical stimulation may have beneficial effects on the regeneration of destructed periodontal tissue in regard of the stimulation of periodontal ligament cells and gingival fibroblasts as well as electrically stimulated bone formation that has been known, and that electrical stimulation may have the potential of clinical application.

• Journal of Periodontal and Implant Science
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• v.28 no.1
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• pp.133-143
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• 1998

Diabetes mellitus is a systemic disease with profound effects on oral health and periodontal wound healing. Uncontrolled diabetes adversely affects surgical wound healing and is often associated with abnormal proliferation of fibroblasts. Human gingival fibroblasts and PDL cells were chosen because they are intimately involved in periodontal therapy and are important for the success of surgical procedure such as guided tissue regeneration. The aim of the present study was to elucidate whether cellular activity and collagen synthesis by glucose pre-treated human gingival fibroblasts and PDL cells are influenced by insulin, and whether healthy cells differ from glucose treated cells. Cells were cultured with DMEM at $37^{\circ}C$, 5% $CO_2$, 100% humidified incubator. To evaluate the effect of glucose on gingival fibroblasts and periodontal ligament cells, the cells were seeded at a cell density of $1{\times}10^4\;cells/well$ culture plates and treated with 20 and 50mM of glucose for 5 days. Then MTT assay was carried out. To evaluate the effect of insulin on glucose-pretreated cells, the cells were seeded at a cell density of $1{\times}10^4\;cells/well$ culture plates and treated with 20 and 50mM of glucose for 5 days. After incubation, $10^3$, $10^4$ and $10^5mU/l$ of insulin were also added to the each well and incubated for 2 days, respectively. Then, MTT assay and collagen synthesis assay were carried out. The results indicate that cellular activity of gingival fibroblasts significantly increased by glucose while periodontal ligament cells were unaffected and cellular activity of gingival fibroblasts and periodontal ligament cells were unaffected by insulin. Collagen synthesis of gingival fibroblast with 20mM glucose and insulin unaffected, but 50mM glucose and insulin increased than control. Collagen synthesis of periodontal ligament cell with 20mM glucose and $10^5mU/l$ insulin significantly increased than other groups and 50mM glucose pretreated PDL cells significantly increased at $10^3mU/l$ insulin but decreased at $10^4mU/l$ insulin. Our findings indicated that these cell types differed in their growth response to glucose, and the increase in collagen synthesis was significantly raised at insulin level of $10^3mU/l$ in gingival fibroblasts and periodontal ligament cells except 20mM glucose pretreated periodontal ligament cells.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.36 no.1
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• pp.7-15
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• 2010

Complex human tissues harbor stem cells and precursor cells, which are responsible for tissue development or repair. Recently, dental tissues such as dental pulp, periodontal ligament (PDL), dental follicle have been identified as easily accessible sources of undifferentiated cells. These tissues contain mesenchymal stem cells that can be differentiate into bone, cartilage, fat or muscle by exposing them to specific growth conditions. In this study, the authors procured the stem cell from pulp, PDL, and dental follicle and differentiate them into osteoblast and examine the bone induction capacity. Dental pulp stem cell (DPSC), periodontal ligament stem cell (PDLSC), and dental follicle precursor cell (DFPC) were obtained from human 3rd molar and cultured. Each cell was analyzed for presence of stem cell by fluorescence activated cell sorter (FACs) against CD44, CD105 and CD34, CD45. Each stem cell was cultured, expanded and grown in an osteogenic culture medium to allow formation of a layer of extracellular bone matrix. Osteogenic pathway was checked by alizarin red staining, alkaline phosphatase (ALP) activity test and RT-PCR for ALP and osteocalcin (OCN) gene expression. According to results from FACs, mesenchymal stem cell existed in pulp, PDL, and dental follicle. As culturing with bone differentiation medium, stem cells were differentiated to osteoblast like cell. Compare with stem cell from pulp, PDL and dental follicle-originated stem cell has more osteogenic effect and it was assumed that the character of donor cell was able to affect on differential potency of stem cell. From this article, we are able to verify the pulp, PDL, and dental follicle from extracted tooth, and these can be a source of osteoblast and stem cell for tissue engineering.

• Journal of Periodontal and Implant Science
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• v.23 no.1
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• pp.97-108
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• 1993

One of the important initial events required for periodontal regeneration is the attachment and subsequent spreading of periodontal ligament cells on the root surface. The purposes of this study is to investigate the attachment and spreading pattern of human periodontal ligament cell on the surface of glass slides. After establishment of a cell line of the primary cell culture from the periodontal ligament of 1st premolar teeth which were extracted for the purpose of orthodontic treatment, author dispersed the cells at $5{\times}10^3\;cells/ml$ into the each 35mm culture petri-dish containing 2 glass slides. To observe the morphological changes of the cells which attached to the surfaces of glasses at every designed time schedule, author used the inverted phase contrast microscope and scanning electron microscope. During the whole experiment culture condition was at $37^{\circ}C$, 100% Humidity, 5% $CO_2$ gas incubator. The following results were obtained. Periodontal ligament cells showed spherical outline and started to attach to glass surface by basal sytoplasmic extension after 10min in culture. After 30min in culture, periodontal ligament cells were attached to glass surface by well - developed filopodia which protruded from the lamellipodia. The cell surface is covered with bubble-like structures and occasional microvillus can be seen with diffculty among these structures. After 1.5hr in culture, peridontal ligament cells shhowed radially well-spread cytoplasm and the nucleus was centered on its cytoplasm. Unspread central region of the cell was covered with numerous microvilli. The change of cell attachment and spreading pattern was manifest at 6hr in culture. At this time, periodontal ligament cell showed elongated outline and an oval-shaped nucleus. After 12hr in culture, periodontal ligament cells showed more stretched fibroblast-like appearance with polarity. Two long lamellipodia can be seen around the both terminal ends of cells. After 24hr in culture, periodontal ligament cells showed spindle shapes and an oval-shaped nucleus was slanted toward one side of the cell.