• Restorative Dentistry and Endodontics
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• 제32권5호
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• pp.419-425
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• 2007

본 연구의 목적은 세 가지 레진계 근관충전실러 (AH 26, EZ fill, AD Seal), 산화아연 유지놀계 근관충전실러(ZOB Seal) 그리고 수산화칼슘계 근관충전실러 (Sealapex)의 방사선 불투과성 및 세포독성을 평가한 것이다. 각 실러를 제조회사의 지시대로 혼합하여 직경 10 mm, 두께 1 mm로 시편을 제작한 후 ISO 6876/2001의 규격에 따라 교합필름을 이용하여 알루미늄 스텝웨지와 함께 방사선 촬영을 시행하였다. 방사선 사진을 디지털화하여 컴퓨터에 저장한 후 Scion image 프로그램을 이용하여 각 단계의 알루미늄 스텝웨지의 두께와 비교하였다. 각 재료의 세포 독성은 불멸화된 인간 치주인대세포 (immortalized human periodontal ligament cell, IPDL)에서 MTT 분석법을 이용하여 시행하였다. EZ fill이 가장 높은 방사선 불투과성을 나타내었고 Sealapex가 가장 낮은 방사선 불투과성을 나타내었다 (p < 0.05). AH 26, AD Seal, ZOB Seal은 중등도의 방사선 불투과성을 나타내었다. Sealapex를 제외한 모든 평가된 재료는 ISO 규격에 부합하는 방사선 불투과성을 보였다. 레진계 실러의 세포독성은 모든 실험 시간대에 걸쳐 다른 계통의 실러에 비해 낮게 나타났다 (p < 0.05) 아울러, EZ fill은 24및 48시간대에서는 AD Seal에 비해, 72 시간대에서는 다른 두 레진계 실러에 비해 높은 세포독성을 보였다. 그러나 레진계 실러에서 방사선 불투과성의 정도와 세포독성과의 관련성은 없었다 (p > 0.05). 이 실험 결과로 볼 때 레진계 실러는 다른 계통의 실러에 비해 방사선 불투과성 면에서 장점을 가지며 생체적합성면에서 우수하다고 사료된다.

• Journal of Periodontal and Implant Science
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• 제31권1호
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• pp.21-41
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• 2001

The goal of Periodontal treatment is predictable periodontal regeneration. But until now, many products including GTR materials and growth factors are beyond of complete regeneration. 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. BMP can promote periodontal regeneration by their ability to stimulate new bone and new cementum formation. But little is known about optimal conditions required for the application. Root conditioning is used for bioacive root change so altered root surface provides a substrate that promotes chemotaxis, migration and attachment of peridontal cells encouraging connective attachment to the denuded root surface. The aim of this study is to investigate whether the acid conditioning change effect of rhBMP-2 on human periodontal ligament cell and osteoblast cell line. 288 periodontally involved root dentin slices are divided into 6 groups, each 48, 1)control, 2)treated with BMP, 3)treated with citric acid 4)treated with citric acid+BMP 5)treated with tetracycline 6)treated with TC+BMP. Each group was devided half, so 12 root dentin slices were seeded with periodontal ligament cells and 12 were seeded with osteoblasts. At day 2 and 7, cell number, protein assay, ALP activitiy was measured. To investigate morphology of cultured cells, SEM was employed. Statistical analysis was performed with SPSS 8.0 either t-test or ANOVA test. The results are ; Protein assay and cell number was slightly decreased in CA+BMP group compared to Ca group but it was not statistically significant and ALP activity was much more increased in CA+BMP group compared to CA group so there was no statistically significance between BMP and CA+BMP group and statistically significant compared to control group. Cell number and protein assay was slightly increased in TC group and ALP activity was much less the BMP group and CA group. Cell number and protein and ALP activity was not much increased in TC+BMP group. TC group and TC+BMP group showed cell morphology change in SEM. This results suggested that application of root surface with citric acid before BMP treatment might give better result in periodontal regeneration.

• Journal of Periodontal and Implant Science
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• 제31권1호
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• pp.109-122
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• 2001

Gingival fibroblasts(GF) and periodontal ligament fibroblasts(PDLF) are the major cellular components of periodontal soft connective tissues, but the precise molecular biological differences between these cells are not yet known. In the present study, we investigated the expression of S100A4, S100A2 calcium-binding protein and osteoblast-specific factor 2(OSF-2, Periostin) mRNA in GF and PDLF in vitro through the process of reverse transcription-polymerase chain reaction(RT-PCR) and Northern blot analysis in each. Human GF and PDLF were isolated from the gingival connective tissue and the middle third of freshly extracted healthy third molars. They were cultured in Dulbecco's Modified Eagle Medium(DMEM) containing 10% fetal bovine serum and cells in the third passage were used in the experiments. After extracting total RNA from cultured cells, RT-PCR and Northern analysis were performed using S100A4-, S100A2- and Periostin-specific oligonucleotide primers and subcloned cDNA probes in each. In PT-PCR and Northern analysis, the expression of S100A4 and Periostin mRNA in GF was slightly detectable. Interestingly, the expression of S100A4 and periostin mRNA in PDLF was much higher than that in GF. On the other hand, S100A2 mPNA was highly expressed in both GF and PDLF. Since there was a marked difference of S100A4 and Periostin expression between GF and PDLF in vitro, these data suggest that S100A4 and periostin could be used as a useful marker for distinguishing cultured gingival fibroblasts and periodontal ligament cells.

• Journal of Periodontal and Implant Science
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• 제29권4호
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• pp.765-785
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• 1999

Bone morphogenetic protein-2/4 (BMP-2/4) are members of Transforming Growth $Factor-{\beta}\;(TGF-{\beta})$ superfamily and they may differentiate the osteoprogenitor cell and induce formation of cartilage and bone in vivo. This study was performed to investigate the effects of bone morphogenetic protein-2/4 on the characteristics of rat periodontal ligament cells(RPDL) and rat calvaria cells(RCV). In the control group, the cells were cultured alone with Dulbeco's Modified Eagle's Medium contained with 20% fetal bovine serum, $100{\mu}/ml$ penicillin, $100{\mu}/ml$ streptomycin. In the experimental groups, recombinant human bone morphogenetic protein-2/4 (25ng, 100ng, 250ng/ml) were added into the above culture condition. And then each group was characterized by examing the cell proliferation at 1, 2, 3, 5, 7th day, the amount of total protein synthesis and alkaline phosphatase activity at 2, 5, 7th day. And also, the calcified nodule was examed. The results were as follows ; 1 . Both RCV and RPDL cells in both control and experimental groups proliferated during the entire experimental period, but there is no stastically significant difference according to the BMP-2/4 concentration. 2 . Amount of total protein synthesis of both cells in both groups was steadily increased until 5th day, but all experimental groups were significantly different from the control group at 7th day. 3. Alkaline phosphatase activity of both cells in both groups was increased during the entire experiment period. In RCV cells, the experimental group treated with 100ng/ml and 250ng/ml BMP-2/4 were significantly different from the control group at 7th day. In RPDL cells, the experimental group treated with 100ng/ml and 250ng/ml BMP-2/4 were significantly different from the control group at 5th day, and all experimental groups were significantly different from the control group at 7th day. 4. In the both of the cultured Rat Periodontal ligament and calvaria cell treated with BMP-2/4 to compared with control group, it revealed more rapid cell polarization, cell aggregation and hyperchromatic stained on HE agent, and even though only 1 day treated with BMP-2/4 both RPDL and RCV showed more rapid cell reaction than control group. More sensivitve cell reaction of RCV were observed than RPDL in this experiment. From the above results, we could conclude that BMP-2/4 influenced the induction, proliferation and differentiation of bone forming cells

• Journal of Periodontal and Implant Science
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• 제32권4호
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• pp.733-744
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• 2002

The fibroblasts are the principal cells in the periodontal ligament of peridontium. As the periodontal ligament fibroblasts (PDLF) show similar phenotype with osteoblasts, the PDLF are thought to play an important role in alveolar bone remodeling. Cell-to-cell contacted signaling is crucial for osteoclast formation. Recently it has been reported that PDLJ enhance the bone resorbing activity of osteoclasts differentiated from hematopoietic preosteoclasts. The aims of this study were to $clarify\;^{1)}$ the mechanism of PDLF-induced osteoclastogenesis $and\;^{2)}$ whether we can use preosteoclast cell line instead of primary hematopoietic preosteoclast cells for studying the mechanism of PDLF-induced osteoclastogenesis. Osteoclastic differentiation of mouse macrophage cell line RAW264.7 was compared with that of mouse bone marrow-derived M-CSF dependent cell (MDBM), a well-known hematopoietic preosteoclast model, by examining, 1) osteoclast-specific gene expression such as calcitonin receptor, M-CSF receptor (c-fms), cathepsin K, receptoractivator nuclear factor kappa B (RANK) ,2) generation of TRAP(+) multinucleated cells (MNCs), and 3) generation of resorption pit on the $OAAS^{TM}$ plate. RAW264.7 cultured in the medium containing of soluble osteoclast differentiation Factor (sODF) showed similar phenotype with MDBM-derived osteoclasts, those are mRNA expression pattern of osteoclast-specific genes, TRAP(+) MNCs generation, and bone resorbing abivity. Formation of resorption pits by osteoclastic MNCs differentiated from sODF-treated RAW264.7, was completely blocked by the addition of osteoprotegerin (OPG), a soluble decoy receptor for ODF, to the sODF-containing culture me야um. The effects of PDLF on differentiation of RAW264.7 into　the TRAP(+) multinucleated osteoclast-like cells were examined using coculture system. PDLF were fxed with paraformaldehyde, followed by coculture with RAW264.7, which induced formation of TRAP(+) MNCs in the absence of additional treatment of sODF. When compared with untreated and fixed PDLF (fPDLF), IL-1 ${\beta}$-treated, or lipopolysaccha-ride-treated and then fixed PDLF showed two-folld increase in the supporting activity of osteoclastogenesis from RAW264.7 coculture system. There were no TRAP(+) MNCs formation in coculture system of RAW264.7 with PDLF of no fixation. These findigs suggested that we can replace the primary hematopoietic preosteoclasts for RAW264. 7 cell line for studying the mechanism of PDLF-induced osteoclastogenesis, and we hypothesize that PDLF control osteoclastogenesis through ODF expression which might be enhanced by inflammatory signals.

• Journal of Periodontal and Implant Science
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• 제30권4호
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• pp.895-913
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• 2000

It is becoming increasingly clear that human gingival fibroblasts(HGF) may play a role in regulating immune responsiveness in inflammatory periodontal lesions. Stimulation of HGF with locally-secreted T cell cytokine $IFN_{\gamma}$ induces human leukocyte antigen class II(HLA II) expression on HGF, which is one of the characteristic feature of professional antigen presenting cells(pAPC). However, $IFN_{\gamma}$-treated HGF and other nonprofessional antigen presenting cells(npAPC) are known to be ineffective or less effective antigen presenter to resting T cells. This study, therefore, was undertaken in an effort to elucidate the differences in expression of cell surface molecules between npAPC in periodontal tissues, such as HGF and periodontal ligament fibroblasts(PDLF), and pAPC such as monocytes/macrophages. Using flow cytometry, the levels of cell surface expression of HLA-D, ICAM-1, LFA-3, and B7-1, which are involved in antigen presentation, were determined in HGF, PDLF and human myelomonocytic cell line THP-1. $IFN_{\gamma}$ clearly induced HLA-D expression on both of fibroblasts and monocytes dose dependently. However, expression level on monocytes were 4 to 5 times higher than that on fibroblasts, and induction rate was faster in monocytes than in fibroblasts. The levels of ICAM-1 expression on fibroblasts and monocytes were enhanced by $IFN_{\gamma}$ in a dose dependent manner. On the other hand, the expression of LFA-3 molecule, which could be detected in fibroblasts and monocytes without cytokine stimulation, was no more enhanced by addition of $IFN_{\gamma}$. B7-1, important costimulatory molecule in T cell activation and proliferation, was not detected on both of fibroblasts and monocytes even when stimulated with $IFN_{\gamma}$, except on monocytes fully differentiated by pretreatment of PMA and treated by $IFN_{\gamma}$. These results suggest that delayed expression of HLA-D and absence of B7-1 on $IFN_{\gamma}$ - treated fibroblasts may at least in part be involved in the ineffectiveness of fibroblasts as primary APC. And it is postulated that although periodontal fibroblasts may not serve as primary APC in normal periodontium, sustained expression of HLA II on ubiquitous fibroblasts in inflammatory lesions may perpetuate immune responses and produce chronic inflammation and tissue injury.

• Journal of Periodontal and Implant Science
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• 제31권1호
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• pp.163-180
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• 2001

The aim of this study was to evaluate the effects of chitosan coating on the attachment, proliferation, functional and morphological change of periodontal ligament cells. Primary human periodontal ligament cells were cultured in dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% antibiotics. In experimental group, cells of 4th to 7th passage were inoculated in the multiwell plates coated with chitosan in concentration of 0.22, 0.2, and $2mg/m{\ell}$. Cell counting and MTT assay were done after 0.5, 1.5, 3, 6 and 24 hours of incubation to evaluate the cell attachment, and then after 2 and 7 days of culture to evaluate the cell proliferation. The alkaline phosphatase activity was measured after 4 and 7 days of culture and the ability to produce mineralized modules was evaluated after 21 days of culture. The results were as follows : 1. The morphology of periodontal ligament cells on the chitosan coating was round or spheric. Round cells were aggregated after 6 hours of culture. Aggregated cells on the chitosan coated surface showed nodule-like appearance after 24 hours of culture and not achieved confluency at 7 days. 2. During early period of culture, the attachment of periodontal ligament cells were inhibited by chitosan coating. Inhibition of cell attachment tended to increase with the concentration of chitosan. 3. At the chitosan concentration of 0.02 and $0.2mg/m{\ell}$, periodontal ligament cells were more rapidly proliferated at 7 days, compared to the control group. At the concentration of $2mg/m{\ell}$, the proliferation of periodontal ligament cells was inhibitied(p<0.01). 4. Alkaline phosphatase activity of periodontal ligament cells was increased in chitosan coated group, especially at the concentration of $0.02mg/m{\ell}$after 4 days of culture.5. Periodontal ligament cells produced mineralized nodules on chitosan coated wells without the addition of mineralized nodule forming materials (ascorbic acid, ${\beta}-glycerophosphat$, dexamethasone). With the addition of mineralized nodule forming materials, periodontal ligament cells produced more mineralized nodules at the concentration of $0.02mg/m{\ell}$, compared to the control. In summary, the attachment, proliferation, cell activity, and alkaline phosphatase activity of periodontal ligament cells depended on the concentration of coated chitosan. Chitosan stimulated mineralized nodule formation by periodontal ligament cells. At the appropriate concentration($0.02mg/m{\ell}$), chitosan could increase alkaline phosphatase activity and stimulate the formation of mineralized nodule by periodontal ligament cells. These results suggest that chitosan can be used as an adjunct for bone graft material, and the matrix of tissue engineering for periodontal regeneration, especially bone regeneration.

• Journal of Periodontal and Implant Science
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• 제31권1호
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• pp.179-193
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• 2001

previous studies have demonstrated an increase in bone mass and density with use of bisphosphonate in osteoporosis. This agent acts as an inhibitor of osteoclastic activity and results in increase of net osteoblastic activity. The purpose of the present study was to examine the effect of the bisphosphonate on osteoblastic activity of the human periodontal ligament cells in vitro. Periodontal ligament cells were primarily obtained from extracted healthy third molars. Cells of 4th to 6th passage were cultured in Dulbecco's modified Eagle's medium containing alendronate sodium or etidronate disodium at the concentration of $10^{-12}{\sim}10^{-6}mol/L$ in 5% $Co_2$ incubator at $37^{\circ}C$. Cell count and MTT assay for cellular activity were done at 2 to 7 days of culture. Alkaline phosphatase activity at 4 to 7 days of culture and formation of mineralized nodules at 28 days of culture with addition of $50{\mu}g/m{\ell}$ ascorbic acid, 10 nM${\beta}-glycerophosphate$, $10^{-7}M$ dexamethasone were evaluated. 1. Alendronate sodium Compared to the control, the proliferation of periodontal ligament cells was generally increased and the cellular activity was maintained at 2 days of culture and generally decreased at 7 days of culture. Alkaline phosphatase activity of periodontal ligament cells was inceased and the formation of mineralized nodules by periodontal ligament cells was enhanced compared to the control. 2. Etidronate disodium The proliferation of periodontal ligament cells was increased at 2 days of culture and decreased or maintained at 7 days of culture. Compared to the control, the cellular activity of periodontal ligament cells was generally decreased. Alkaline phosphatase activity of peridontal ligament cells was increased and the formation of mineralized nodules by periodontal ligament cells was enhanced compared to the control. These results suggest that alendronate sodium and etidronate disodium may have a potential effect on osteoblastic lineage of periodontal ligament cells, distinct from their inhibitory action on osteoclasts and could contribute to enhance periodontal regeneration and alveolar bone regeneration.

• Journal of Periodontal and Implant Science
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• 제27권4호
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• pp.923-939
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• 1997

This study was performed to evaluate the effect of mixed culture of rat's calvaria cells and periodontal ligament cells on calcification. These cells have been known to do important role on the periodontal tissue regeneration, especially alveolar bone and cementum. Experimental groups were made which based on the different rate of rat's calvaria cells and periodontal ligament cells, and then these cells were cultured with Dulbecco's Modified Eagle's Medium contained with 10% fetal bovine serum, $50{\mu}g/ml$ ascorbic acid, and 10mM/ ml $Na-{\beta}-glycerophosphate$. Each group was characterized by examining the cell proliferation rate, amount of total protein synthesis, alkaline phosphatase activity, and the number of calcified nodules in vitro. In cell proliferation rate , the cells of control groups were cultured Dulbecco's Modified Eagle's Medium contained with 10 % fetal bovine serum. The results were as follows : 1. The cell proliferation rate in control groups decreased stastically significantly along with the decrease of the rate of bone cells at 7 day and 20 day(P < 0.01). 2. The cell proliferation rate in experimental groups decreased stastically significantly along with decrease of the rate of bone cells at 3 day and 14 day(P < 0.01). 3. The amount of total protein synthesis was significantly decreased along with decrease of the rate of bone cells at 3 day and 6 day(p < 0.01). 4. Alkaline phosphatase activity showed reverse time dependent pattern and was significantly decreased along with decrease of the rate of bone cells during the experimental periods (P < 0.01). 5. Calcified nodules were observed in group 1 (Rat calvaria cells alone) for the first time, and the number of calcified nodule decreased stastically significantly along with the decrease of the rate of bone cells at 12 day(P < 0.01). From the above results, When bone cells and periodontal ligament cells were mixed cultured, the cell proliferation rate was mostly dependent on the actual rate of bone cells and same pattern was showed in amount of total protein synthesis, alkalinephosphatase activity, and the number of calcified nodules. And the calcified nodule forming capacity of bone cells was inhibited by periodontal ligament cells

• Journal of Periodontal and Implant Science
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• 제35권1호
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• pp.99-109
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• 2005

It has been focused on the importance of the host inflammatory response in periodontal pathogenesis and progression, treatment has been introduced to control the host response and the method, which diminishes production and activity of MMP by doxycycline, has been used in periodontal field. MMP is a proteolytic enzyme which plays a major role in tissue destruction and MMP-1 is secreted in the periodontally healthy tissue, while MMP-8, 9, 13, etc in the inflammatory state. Among these, MMP-13 has been discovered lately and reported to degrade primarily type II collagen. Periodontal ligament (PDL) cell plays a role in destruction of periodontal tissue. This study was to evaluate the effect of doxycycline and mefenamic acid, non-steroidal antiinflammatory drug on MMP-13 mRNA expression in the rat PDL cell. Doxycycline concentration of $1{\sim}100\;{\mu}g/ml$ was added rat PDL cell and cell activity was measured by MIT assay at day 1 and 3. MMP-13 gene expression was evaluated by RT-PCR after PDL cells were pre-treated for 1hour with doxycycline (50 ${\mu}g/ml$) alone or with mefenamic acid ($10^{-6}M$), then added $IL-1{\beta}$(1.0 ng/ml) and incubated for 16-18 hours. The results are as follows: 1. Cell activity decreased Significantly at 24 and 72 hours in 100 ${\mu}g/ml$ (p<0.05). 2. Level of MMP-13 mRNA was in 20.2% increase by $IL-1{\beta}$ and in pre-treating doxycycline group, expression of $IL-1{\beta}$ induced MMP-13 mRNA was inhibited by 31% than $IL-1{\beta}$ treated only. 3. Mefenamic acid did not inhibit on the expression of $IL-1{\beta}$ induced MMP-13 mRNA, while mefenamic acid in combination with doxycycline inhibited the expression by 41% compared to only $IL-1{\beta}$ stimulation. These results suggest that doxycycline synergistically inhibit the expression of $IL-1{\beta}$ induced MMP-13 mRNA in combination with mefenamic acid.