• Journal of Periodontal and Implant Science
• /
• v.32 no.3
• /
• pp.445-456
• /
• 2002

The purpose of this study were to determine that dexamethasone(Dex) induces differentiation of periodontal ligament(PDL) cells to osteoblastic cells and to investigate expression of matrix Gla protein(MGP), which is one of bone matrix protein. The isolated human PDL cells and gingival fibroblasts were prepared and cultured. The fourth or sixth sub-passage cells were used in this experiments. control group, ascorbic acid and ${\beta}$-glycerophosphate treated group, ascorbic acid, ${\beta}$-glycerophosphate and l00nM Dex treated group, ascorbic acid, ${\beta}$-glycerophosphate, and 5 ${\mu}M$ Dex treated group were made for study. The results were as follows: Cellular morphological change of PDL cells according to time was investigated. At first, the cells exhibited confluent monolayer of spindle or polygonal appearance. The multilayer of cells were seen after 7 days of treatment. After 14 days, the cells lost polarity and were densely packed. The mineralized nodule formation was seen at 21 days in the only Dex treated PDL cell groups. In the gingival fibroblast groups and no Dex treated PDL cell groups, the mineralized nodule was not seen. The mineralized nodule formation of 5 ${\mu}M$ Dex treated group was higher than 100 nM Dex treated group. Alkaline phosphatase(ALP) activity was higher in the Dex treated PDL cell groups of 14 and 21 days than 0 and 7 days. MGP was expressed in the control and all experimental groups and the expression was constant at 0,7,14,21 day. The above results confirm that Dex is affected to differentiation of the PDL cells to osteoblastic or cementoblastic cells and has dose-dependent effect for mineralization. And, MGP is expressed in the PDL cells and is not affected to mineralization of PDL cells.

• Journal of Periodontal and Implant Science
• /
• v.25 no.1
• /
• pp.45-55
• /
• 1995

치주질환의 치유에 있어서 치주인대 세포의 증식과 분화는 매우 중요하다. 몇몇 학자들에 의해 치주인대 세포의 증식과 분화에 영향을 주는 platelet derived growth나 fibronectin과 같은 growth factor에 대한 연구가 있었다. 이 연구는 홍삼 총사포닌이 치주인대 세포에 미치는 세포독성과 세포의 성장 및 분화에 미치는 영향을 규명하고자 사람의 치주인대 세포를 분리, 배양하여 실험하였다. 총사포닌이 치주인대 세포에 미치는 세포독성을 특정하기 위해 여러가지 농도의 총사포닌을 세포배양액에 첨가하여 1주일 배양후의 결과와 단일 농도($1{\mu}g/ml$})하에서의 세포 성장을 혈구계산반을 사용하여 관찰하였다. 치주인대 세포가 조골세포양세포로의 분화과정에 사포닌이 영향을 미치는 것을 관찰하기 위해 개개의 총사포닌 농도)0.1,1,$10{\mu}g/ml}$)를 세포배양액에 첨가하여 배양하였다. 치주인대 세포가 조골세포의 표현형으로 분화되는데 미치는 총사포닌의 영향을 알아보기 위하여 총사포닌의 단일 농도($1{\mu}g/ml$)하에서 $50{\mu}g/ml$ ascorbic acid와 10mM ${\beta}-glycerophosphate$를 배양액에 첨가하여 배양후 von Kossa's staining을 시행하여 생성된 골결절을 관찰하였다. 이상의 실험에서 얻어진 결과는 아래와 같다. 1. 각각의 농도를 투여한 결과, $1{\mu}g/ml$의 총사포닌에 의해서 세포독성이 유의성있게 증가하였다. 2. 0.01,0.01,1,$10{\mu}g/ml$의 총사포닌을 세포배양액에 첨가한 다음 7일 후의 고나찰시 cell viability가 실험농도 모두에서 유의성있게 증가하였다. 3. 0.1,1,10,$100{\mu}g/ml$의 농도에서 유외성있는 세포 증식이 있었다. 4. $1{\mu}g/ml$의 총사포닌을 세포배양액에 첨가한 다음 1,3,5,7,9일의 관찰시 시간경과에 따라 유의성있는 세포 증식이 있었다. 5. $10{\mu}g/ml$의 총사포닌을 세포배양액에 첨가시 ALP activity가 대조군에 비해 유외성있게 증가하였다. 6. $1{\mu}g/ml$의 총사포닌으로 배양된 치주인대 세포내에서 ALP positive cell이 관찰되었다. 7. $1{\mu}g/ml$의 총사포닌으로 배양된 치주인대 세포내에서 골결절 형성이 관찰되었다.

• The korean journal of orthodontics
• /
• v.43 no.6
• /
• pp.294-301
• /
• 2013

Objective: To determine the interleukin (IL)-6 levels in gingival crevicular fluid (GCF) of patients with severe root resorption after orthodontic treatment and investigate the effects of different static compressive forces (CFs) on IL-6 production by human periodontal ligament (hPDL) cells and the influence of IL-6 on osteoclastic activation from human osteoclastic precursor (hOCP) cells in vitro. Methods: IL-6 levels in GCF samples collected from 20 patients (15 and 5 subjects without and with radiographic evidence of severe root resorption, respectively) who had undergone orthodontic treatment were measured by ELISA. The levels of IL-6 mRNA in hPDL cells and IL-6 protein in conditioned medium after the application of different uniform CFs (0, 1.0, 2.0, or 4.0 $g/cm^2$ for up to 72 h) were measured by real-time PCR and ELISA, respectively. Finally, the influence of IL-6 on mature osteoclasts was investigated by using hOCP cells on dentin slices in a pit-formation assay. Results: Clinically, the IL-6 levels were significantly higher in the resorption group than in the control group. In vitro, IL-6 mRNA expression significantly increased with increasing CF. IL-6 protein secretion also increased in a time- and magnitude-dependent manner. Resorbed areas on dentin slices were significantly greater in the recombinant human IL-6-treated group and group cultured in hPDL cell-conditioned medium with CF application (4.0 $g/cm^2$) than in the group cultured in hPDL cell-conditioned medium without CF application. Conclusions: IL-6 may play an important role in inducing or facilitating orthodontically induced inflammatory root resorption.

• The korean journal of orthodontics
• /
• v.31 no.6 s.89
• /
• pp.589-600
• /
• 2001

Insulin-like growth factor I (IGF-I) has the local tissue regulating actions. In bone, IGF-I increases the replication of osteoblastic lineage, probably preosteoblasts, and enhances osteoblastic collagen synthesis and matrix composition rates. The purpose of this study was to investigate the local regulatory effect of IGF-I on periodontium totally, both in an autocrine and paracrine manner. To examine the effect of IGF-I directly on osteoblast (OB) of test rats, and indirectlv on OB via periodontal ligament fibroblast (PDLF), and the effect of gingival fibroblast (GF) on OB via cellular paracrine manner for the understanding of humoral action of adjacent tissue, GF and PDLF were obtained from male Sprague-Dawley rats of six to eight weeks of age. OB was obtained iron frontal and parietal calvarial bone of Sprague-Dawley 21day-old-fetus. After each tell was Incubated 24 hours, for collecting conditioned medium, different concentrations of IGF-I (1,10,100 ng/ml,1ml/well) was adding in the GF, PDLF cells, and the supernatant from these cultures was put into the primary OB culture with $1{\times}10^4$cell/ml/well. The experimental group was divided into six groups control OB, IGF-I treated OB, OB culture with conditioned medium from PDLF, OB culture with conditioned medium from IGF-I treated PDLF, OB culture with conditioned medium from GF, OB culture with conditioned medium from IGF-I treated GF. After final IGF-I treatment, OB was Incubated for 24 hours, and alkaline phosphatase activity assay, BMP expression, cell proliferation measurement using MTT assay, total protein measurement, Collagen synthesis assay using western blot, and examination of bone nodule synthesis were done. Alkaline phosphatase expressions were increased in the group of PDLF-IGF-I supernatant treatment. Direct IGF-I treatment with concentrations of 100ng/m1 showed increased viable tell number measured by MTT assay. And IGF-I treatment did not increase total protein amount. The entire experimental group showed BMP2, 4 expression in western blot, and there was no significant difference between control and experimental groups. These results suggested that supernatant from PDLF effects on increasing cellular activities of OB regardless of IGF-I, and at high concentration, IGF-I increases OB tell proliferation.

• Journal of Periodontal and Implant Science
• /
• v.23 no.2
• /
• pp.261-281
• /
• 1993

The author transplanted periodontally-diseased teeth which had been treated with citric acid into a clinically healthy extraction sockets and periodontally-affected extraction sockets, and compared with the healing processes within these tissues. Recipient sites were prepared by surgically removing a part of alveolar bone of premolars of adults dogs, placing elastic orthodontic ligatures for 8weeks, thereby inducing periodontal disease. The diseased roots were extracted and transplanted into healthy extraction sockets, and these were designated as control group 1. Diseased roots transplanted into diseased sockets were designated as control group 2. Diseased roots which had been root planed, treated with citric acid and transplanted into healthy sockets were designated as experimental group 1, while identically treated roots which had been transplanted into diseased sockets were designated as experimental group 2. Observations were made at weeks 2, 8 and 12, with following results. 1. At week 2, experimental group 2 showed some inflammatory cell infiltration in the connective tissue above the extraction sockets, while control groups showed less inflammatory or foreign body reactions throughout the experiment. 2. In both control groups, root surface resorption was observed throughout the experiment, while experimental groups showed a little resorption. 3. Control group 1 & 2 showed ankylosis by newly-formed bone ground the resorbed root surfaces, while experimental group 1 & 2 displayed collagen fibers which are not functionally-arranged, with random, loose arrangement or parallel orientation to root surfaces, and newly-formed bone outside of them. 4. In both control groups & experimental groups which had been transplanted into a clinically healthy extraction sockets & periodontally affected extraction sockets groups, histological differences were not significant. 5. Root resorption or ankylosis in control group 1 & 2 had increased quantitatively as experiment progressed. 6. New bone formation developed from the base and lateral wall of extraction sockets. In both control groups & experimental groups, root surfaces lying next to the upper portion of extraction sockets showed little alveolar bone formation and surrounded by connective tissue fiber at weeks 2 & 8, while at weeks 12, they did show alveolar bone formation. 7. At week 12, experimental group 2 showed numerous cells which appeared to be periodontal ligament cells, with functionally arranged connective tissue fibers between the roots and alveolar bone.

• Journal of Periodontal and Implant Science
• /
• v.28 no.4
• /
• pp.545-559
• /
• 1998

Magnoliae cortex has been used as a drug for treatment of fractures in Chinese medicine and safflower(Carthamus tinctorius $Linn{\acute{e}}$) has been traditionally used for treatment of blood stasis. The purpose of present study was to examine the biologic effects of magnoliae cortex extract and safflower extract mixture(MSM) on human periodontal ligament cells and fetal rat calvarial osteoblasts and on healing of rat calvarial defects. The ethanolic extracts of magnoliae cortex(MCE), safflower seed(SSE), Zea May L(ZML) were prepared as positive control group. MSM mixed to the ratios of 1 : 1, 1 : 2, 1 : 5 and 1 : 10 were used as test group. The effects of each agents on the growth and survival, ALPase activity, cell proliferation and tissue regenerative effect of each extracts was evaluated by histomorphometric measuring of newly formed bone on the 8 mm defect in rat calvaria after oral administration of 2 ratio groups(1 : 5 and 1 : 10) at 3 different doses (0.1, 0.25 and 0.5g/kg per day). MSM stimulated the growth and survival rate of osteoblasts and PDL cells more than any other agents. The growth and survival rate were increased as the proportion of safflower seed extract was increased. MCE, SSE, ZML stimulated the ALPase activity of osteoblast and PDL cell in comparison to the negative control group. But all groups of MSM regardless of ratio of safflower seed extract stimulated the ALPase activity than any other agent. The ALPase activity was also increased as the proportion of safflower seed extract was increased. Although MCE, SSE, ZML stimulated the proliferation of osteoblasts. 1 : 5 and 1 : 10 ratio MSM showed significant increase in stimulation of proliferation of osteoblasts. No agent significantly increased proliferation of PDL cells. Significant new bone formation were seen where 1 : 5 ratio, 0.5g/kg group and 1 : 10 ratio, 0.25, 0.5g/kg groups were used. These results show that magnoliae cortex extract and safflower seed extract mixture can potentially increase bone regeneration ability.

• Journal of Periodontal and Implant Science
• /
• v.32 no.1
• /
• pp.1-12
• /
• 2002

The periodontal ligament(PDL) is a unique tissue that is crucial for tooth function. However, little is known of the molecular mechanisms controlling PDL function. PDL-specific protein;PDLs22 had been previously identified as a novel protein isolated from cultured human PDL fibroblasts using subtraction hybridization between human gingival fibroblasts and PDL fibroblasts. The aim of this study was to examine the expression pattern and tissue localization of PDLs22 protein in embryonic and various postnatal stages of developing mouse using immunohistochemical staining. Embryos (E18) and postnatal (P1, P4, P5, P15, P18) were decapitated and the heads were fixed overnight in a freshly prepared solution of 4% paraformaldehyde. Some specimens were decalcified for $2{\sim}4$ weeks in a solution containing 10% of the disodium salt of ethylenediamine-tetraacetic acid (EDTA). Next, tissues were dehydrated, embedded in paraffin and sectioned serially at $6{\mu}m$ in thickness. Polyclonal antiserum raised against PDLs22 peptides, ISNKYLVKRQSRD, were made. The localization of PDLs22 in tissues was detected by polyclonal antibody against PDLs22 by means of immunohistochemical staining. The results were as follows; 1. Expression of PDLs22 protein was not detected in the tooth germ of bud and cap stage. 2. At the late bell stage and root formation stage, strong expression of PDLs22 protein was observed in developing tooth follicle, osteoblast-like cells, and subodontoblastic cells in the tooth pulp, but not in gingival fibroblasts, ameloblasts and odontoblasts of tooth germ 3. In erupted tooth, PDLs22 protein was intensely expressed in PDL and osteoblast-like cells of alveolar bone, but not in gingival fibroblasts, mature osteocytes and adjacent salivary glands. 4. In the developing alveolar bone and mid-palatal suture, expression of PDLs22 protein was seen in undifferentiated mesenchymal cells and osteoblast-like cells of developing mid-palatal suture, but not in mature osteocytes and chondrocytes. These results suggest that PDLs22 protein may play an important role in the differentiation of undifferentiated mesenchymal cells in the bone marrow and PDL cells, which can differentiate into multiple cell types including osteoblasts, cementoblasts, and PDL fibroblasts. However, more researches should be performed to gain a better understanding of the exact function of PDLs22 protein which related to the PDL cell differentiation.

• Restorative Dentistry and Endodontics
• /
• v.28 no.5
• /
• pp.385-391
• /
• 2003

The purpose of this study is to examine the viability of PDL cells in rat molars by using MTT assay and to verify the MTT assay through the histologic observation. Thirty of Sprague-Dawley white female rats of 4-weeks old with a body weight of about 100 grams were used. Groupings are as follows : Immediate Group : Positive control group(n=10)-after extraction immediately. Dried Group : Negative control group(n=10)-after drying for an hour under warm dry. $ViaSpan^{\circledR}$ Group : 1hour $ViaSpan^{\circledR}$ group(n=10)-after storing in $ViaSpan^{\circledR}{\;}at{\;}4^{\circ}C$ for 1hour. Ten teeth of each group were treated as same as above and replanted to the original socket of experimental animals. After two weeks of replantation. all the experimental animals were sacrificed. And after fixation, extracted maxillary jaw was dimineralized. After it was embedded in paraffin. serial section by $5\mu\textrm{m}$ was carried out and for construction of specimen, hematoxylin-eosin dye was used. The mean MTT measurement of immediate group(positive control) is 2.81 and the mean measurement of dried group(negative control) is 0.98 which is significantt differnt(P<0.05), The mean measurement of $ViaSpan^{\circledR}$ group is 2.65 and there is significant difference between dried group and $ViaSpan^{\circledR}$ group(P<0.05), However, there is no difference between immediate group and $ViaSpan^{\circledR}$ group. The average resorption points of immediate group is 3.03 points. In the dried group, average 6.44 points resorption and 2.68 points showed resorption in the $ViaSpan^{\circledR}$ group. Unlike with MTT assay, there was no significant difference between the immediate group and $ViaSpan^{\circledR}$ group. The usage of MTT assay as a viable cell marker may give us a better indication of the maintenance of periodontal ligament cell vitality.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• v.39 no.2
• /
• pp.55-62
• /
• 2013

Bone tissue engineering is one of the important therapeutic approaches to the regeneration of bones in the entire field of regeneration medicine. Mesenchymal stem cells (MSCs) are actively discussed as material for bone tissue engineering due to their ability to differentiate into autologous bone. MSCs are able to differentiate into different lineages: osteo/odontogenic, adipogenic, and neurogenic. The tissue of origin for MSCs defines them as bone marrow-derived stem cells, adipose tissue-derived stem cells, and, among many others, dental stem cells. According to the tissue of origin, DSCs are further stratified into dental pulp stem cells, periodontal ligament stem cells, stem cells from apical papilla, stem cells from human exfoliated deciduous teeth, dental follicle precursor cells, and dental papilla cells. There are numerous in vitro/in vivo reports suggesting successful mineralization potential or osteo/odontogenic ability of MSCs. Still, there is further need for the optimization of MSCs-based tissue engineering methods, and the introduction of genes related to osteo/odontogenic differentiation into MSCs might aid in the process. In this review, articles that reported enhanced osteo/odontogenic differentiation with gene introduction into MSCs will be discussed to provide a background for successful bone tissue engineering using MSCs with artificially introduced genes.

• International Journal of Oral Biology
• /
• v.36 no.2
• /
• pp.91-101
• /
• 2011

MspTL is the major surface protein of Treponema lecithinolyticum associated with periodontitis and endodontic infections. Our recent investigation revealed that MspTL induces proinflammatory cytokines and intercellular adhesion molecule 1 in THP-1 cells and periodontal ligament cells. In this study we conducted oligonucleotide microarray analysis to investigate the global transcriptional regulation in THP-1 cells stimulated with purified recombinant MspTL. MspTL upregulated the expression of 90 genes in THP-1 cells at least four fold, and the functions of these genes were categorized into adhesion, apoptosis/antiapoptosis, cell cycle/growth/differentiation, chemotaxis, cytoskeleton organization, immune response, molecular metabolism, proteolysis, signaling, and transcription. The majority of the modified genes are known to be NF-${\kappa}B$-responsive and interferon-stimulated genes (ISGs). The expression of 12 selected genes was confirmed by real-time RT-PCR. Because prostaglandin $E_2(PGE_2)$ is an important inflammatory mediator and Cox-2 was found to be induced by MspTL in the microarray analysis, we determined the level of $PGE_2$ in the culture supernatants of MspTL-treated cells and found that MspTL significantly increased $PGE_2$. Our results provide insight into the gene regulation of host cells in response to MspTL, and may contribute to the understanding of the molecular mechanism in periodontitis.