This study evaluated cell viability and cytokine release in immortalized human oral fibroblasts (hTERT-hNOFs) and keratinocytes (IHOK) exposed to a dental-impregnated gingival retraction cord. To prepare the extracts, dental gingival retraction cords impregnated with aluminum chloride hexahydrate were immersed in a cell culture medium for 24 h at 37 °C. hTERT-hNOFs and IHOK were cultured for 24 h. The cell culture medium was removed and extracts of the dental gingival retraction cords were added. After incubation with the extract solution, cell viability was evaluated using an MTT assay. The levels of the cytokines IL-1α and IL-8 were measured in the supernatants of each cell type. The cell viability after exposure to the extract solution for 10 min exceeded 70 % in both cell types. The ET50 values for hTERT-hNOF and IHOK were 35.75 and 28.98 min, respectively. For IHOK, the IL-1α level was (5.35 ± 5.22) pg/mL at 10 min, (3.58 ± 5.38) pg/mL at 20 min, and (2.85 ± 4.28) pg/mL at 60 min of exposure (p > 0.05). The IL-8 level in IHOK was (67.16 ± 18.70) pg/mL at 10 min, (78.36 ± 7.50) pg/mL at 20 min, and (111.9 ± 26.10) pg/mL at 60 min of exposure (p > 0.05). Cytokine release was not observed from hTERThNOFs. Based on these results, cell viability and cytokine release were confirmed in cells exposed to the impregnated gingival retraction cord. In addition, the application of the extracts to hTERT-hNOF and IHOK during the actual contact time and determination of ET50 may be beneficial for evaluating the biocompatibility of dental-impregnated gingival retraction cords.

In the present study, we investigated the effect of staurosporine on the formation of cellular processes in human gingival fibroblasts and rat astrocytes. Staurosporine caused a rapid induction of process formation in human gingival fibroblasts and rat astrocytes in a concentration dependent manner. The process formation of human gingival fibroblasts and rat astrocytes was prevented by the pretreatment with N-acetylcysteine, suggesting that staurosporine-induced ROS production was responsible for the process formation. Colchicine, a microtubule depolymerizing agent, inhibited the staurosporine-induced process formation, whereas cytochalasin D, an actin filament breakdown agent, failed to suppress the formation of cellular processes. This result indicated that polymerization of microtubule, and not actin filament, was responsible for the formation of cellular processes induced by staurosporine. In support of this hypothesis, Western blot analysis was conducted using anti-tubulin antibody, and the results showed that the amount of polymerized microtubule was increased by the treatment with staurosporine while that of depolymerized beta-tubulin in soluble fraction was decreased. These results indicate that staurosporine induces ROSmediated, microtubule-dependent formation of cellular processes in human gingival fibroblasts and rat astrocytes.

Leptin is one of the adipocytokines produced from adi- pose tissue but its functions in periodontal tissue have not previously been investigated. In our current study, we exa- mined the effects of leptin on the expression of interleukin (IL)-6 and IL-8 in periodontal ligament (PDL) cells and gingival fibroblasts. Leptin receptor expression was evalua- ted by RT-PCR and the production of cytokines was mea- sured by ELISA. The phosphorylation of Akt and Erk1/2 was assessed by western blotting. mRNA of long and short form leptin receptors were detected in both PDL cells and gingival fibroblasts. Leptin was found to increase the pro- duction of IL-6 and IL-8 in both of these cell types, an effect which was not blocked by polymyxin B, an inhibitor of lipopolysaccharide (LPS). Leptin did not alter the pro- duction of IL-6 and IL-8 induced by LPS in PDL cells but increased Akt and Erk1/2 phosphorylation in these cells. These results suggest that leptin acts as an inducer of IL-6 and IL-8 in PDL cells and gingival fibroblasts.

Retinoic acid plays an important role in the regulation of cell growth and differentiation. In our present study, we evaluated the effects of all-trans retinoic acid (RA) on cell proliferation and on the cell cycle regulation of human gingival fibroblasts (HGFs). Cell proliferation was assessed using the MTT assay. Cell cycle analysis was performed by flow cytometry, and cell cycle regulatory proteins were determined by western blot. Cell proliferation was increased in the presence of a 0.1 nM to 1μM RA dose range, and maximal growth stimulation was observed in cells exposed to 1 nM of RA. Exposure of HGFs to 1 nM of RA resulted in an augmented cell cycle progression. To elucidate the molecular mechanisms underlying cell cycle regulation by RA, we measured the intracellular levels of major cell cycle regulatory proteins. The levels of cyclin E and cyclin-dependent kinase (CDK) 2 were found to be increased in HGFs following 1 nM of RA treatment. However, the levels of cyclin D, CDK 4, and CDK 6 were unchanged under these conditions. Also after exposure to 1 nM of RA, the protein levels of p21 WAF1/CIP1 and p16 INK4A were decreased in HGFs compared with the control group, but the levels of p53 and pRb were similar between treated and untreated cells. These results suggest that RA increases cell proliferation and cell cycle progression in HGFs via increased cellular levels of cyclin E and CDK 2, and decreased cellular levels of p21 WAF1/CIP1 and p16 INK4A.

The pattern of wound healing process differs markedly according to the cell types. Gingival wounds heal more rapidly without scar, however dermal wounds show collagen laid down in thick disorganized patterns and keloid formation. This h as b een s uggested t o be d ue t o the presence of d ifferent E C M components a nd c ytokines a s well a s growth factors. The purpose of this study was to examine the differential expression of genes in connection with keloid formation in gingival fibroblasts (hGFs) and dermal fibroblasts (hDFs) in response to inflammation. In this study, we investigated the differences between hGFs and hDFs in the expression and production of cyclooxygenase (COX-2), prostaglandins E2 (PGE2), transforming growth factor (TGF)-β, collagens, matrix metalloproteinases (MMPs), and tissue inhibitors of matrix metalloproteinases (TIMPs) which play important roles in collagen deposition in wound healing. The hGFs and hDFs were primary cultured and allocated to arachidonic acid (AA) treatment group and control group. Protein and mRNA were extracted right after (0 hr) and 24 hr after AA treatment. At a defined concentration of AA in hGFs and hDFs, MTT assay was performed. The mRNA and protein expression levels of COX-2, TGF-β, collagen 1 and 3, MMP 1 and TIMP 1 were examined by Real-time PCR and Western blots. The amounts of PGE2 were measured by enzyme-linked immunosorbent assay (ELISA).The expression of COX-2 and TGF-β exhibited reduced levels in hGFs , but were increased in hDFs at 24 hr after AA treatment. Production of PGE2 was increased in hGFs and hDFs at right after AA treatment but, not changed at 24 hr after AA treatment. The protein and mRNA expression of collagen 1 and 3 were decreased in hGFs , whereas increased in hDFs at 24 hr AA treatment. Expression of MMP-1 protein was increased in hGFs at 24 hr but, was decreased in hDFs at 24 hr compared with that of control. The protein expression of TIMP-1 was decreased in hGFs but, was increased in hDFs at 24 hr compared with that of control. These observations demonstrate differential expression between gingival and dermal fibroblasts in regulation of collagenolytic capacity by extracellular matrix-associated genes in keloid formation associated with wound repair.

Periodontal disease is a major oral disorder and comprises a group of infections that lead to inflammation of the gingiva and the destruction of periodontal tissues. PPARy plays an important role in the regulation of several metabolic pathways and has recently been implicated in inflammatory response pathways. However, its effects on periodontal inflammation have yet to be clarified. In our current study, we evaluated the anti-inflammatory effects of PPARy on periodontal disease. Human gingival fibroblasts (HGFs) treated with lipopolysaccharide (LPS) showed high levels of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), matrix metalloproteinase-2 (MMP-2), and -9 (MMP-9). Moreover, these cells also showed upregulated activities for extracellular signal regulated kinase (ERK1/2), inducible nitric oxide synthase (iNOS) and cyclooxygnase-2. However, cells treated with Ad/PPARy and rosiglitazone in same culture system showed reduced ICAM-1, VCAM-1, MMP-2, -9 and COX-2. Finally, the anti-inflammatory effects of PPARy appear to be mediated via the suppression of the ERK1/2 pathway and consequent inhibition of NF-kB translocation. Our present findings thus suggest that PPARy indeed has a pivotal role in gingival inflammation and may be a putative molecular target for future therapeutic strategies to control chronic periodontal disease.

This study examined the effects of red light generated from a light emitting diode (LED) upon proliferation and mitochondrial stress in human gingival fibroblasts (hGFs). Cells were exposed to LED-generated red light at a clinically relevant intensity and distance with a 610-630 nm wavelength for various times (0-48 min). At different exposure times, cells were processed for the analysis of succinate dehydrogenase (SDH) activity, proliferation, mitochondrial membrane potential (MMP) and cytotoxicity. Cell cycle progression was also investigated by flow cytometry after staining with propidium iodide. Red light exposure was found to inhibit SDH activity and DNA synthesis in hGFs in a time-dependent manner. Light exposure also reduced the MMP levels in these cells and this was closely associated with a G0/G1 arrest. In contrast, exposure of hGFs to red light for 48 min led to a dramatic loss of MMP with an attendant increase in cytotoxicity. These findings demonstrate that LED-generated red light may cause mitochondrial stress and growth inhibition in hGFs during tooth whitening therapy, depending on the length of the exposure.

Nitric oxide (NO) acts as an intracellular messenger at the physiological level but can be cytotoxic at high concentrations. The cells within periodontal tissues, such as gingival and periodontal fibroblasts, contain nitric oxide syntheses and produce high concentrations of NO when exposed to bacterial lipopolysaccharides and cytokines. However, the cellular mechanisms underlying NO-induced cytotoxicity in periodontal tissues are unclear at present. In our current study, we examined the NO-induced cytotoxic mechanisms in human gingival fibroblasts (HGF). Cell viability and the levels of reactive oxygen species (ROS) were determined using a MTT assay and a fluorescent spectrometer, respectively. The morphological changes in the cells were examined by Diff-Quick staining. Expression of the Bcl-2 family and Fas was determined by RT-PCR or western blotting. The activity of caspase-3, -8 and -9 was assessed using a spectrophotometer. Sodium nitroprusside (SNP), a NO donor, decreased the cell viability of the HGF cells in a dose- and time-dependent manner. SNP enhanced the production of ROS, which was ameliorated by NAC, a free radical scavenger. ODQ, a soluble guanylate cyclase inhibitor, did not block the SNP-induced decrease in cell viability. SNP also caused apoptotic morphological changes, including cell shrinkage, chromatin condensation, and DNA fragmentation. The expression of Bax, a member of the proapoptotic Bcl-2 family, was upregulated in the SNP-treated HGF cells, whereas the expression of Bcl-2, a member of the anti-apoptotic Bcl-2 family, was downregulated. SNP augmented the release of cytochrome c from the mitochondria into the cytosol and enhanced the activity of caspase-8, -9, and -3. SNP also upregulated Fas, a component of the death receptor assembly. These results suggest that NO induces apoptosis in human gingival fibroblast via ROS and the Bcl-2 family through both mitochondrial- and death receptor-mediated pathways. Our data also indicate that the cyclic GMP pathway is not involved in NO-induced apoptosis.

The purpose of the present study is to investigate the optimal wavelength, frequency and energy density for set up the photobiologic treatment of periodontal disease. To establish the present study, λ scan of 500㎚ to 900㎚ was used to search the optimal wavelength for maximal proliferation of human gingival fibroblasts. Cell proliferation assay was carried out as MTT assay. Light intensity of 0.8 to 3.25mW, frequency of 0 to 584㎐ and 0 to 2hours was applied for investigation of optimal energy density, frequency and applied duration. Finally, 628㎚ with 1mW/cm2 for 1hour of LED irradiation resulted in maximal proliferation of gingival fibroblasts. These results suggest that LED irradiation on gingival fibroblast show different proliferation according to the condition of irradiation, and demonstrate that LED irradiation can control the quantity of cell proliferation.

Human gingival fibroblasts are necessary for oral homeostaslS These cells are fundamental in tissue healing and tissuc remodeling processes under a response to physiological actions such as mastication, Collagen and elastin, that are extracell ular glycoprotein of gingival fibroblast, are found in all animals, '1γpe 1 collagen is most dominant protein found in human gingival fibroblasts , Matrix metalloproteinase-1(MMP-1) has a role play in destruction of metabolism of extracellular matrix(ECM) and MMP-1 can destroy many ECMs as well as non-ECM molecules MMP-1’s local activation is conytolled by tissue inhibitor of metalloproteinase-1(TIMP-1) , Therefore, it is important to have a balance between in both s ituations MMPs and TIMPs of increased 0 1' decreased extracelluar matrix molecules, The purpose of trus study is to find out the effect of physical stimulus to human gingival fibroblast on mRNA, proteins of collagen 1, elastin, MMP-1, and TIMP-1 Healthy human gingival fibroblasts were separated and cultur때 in DMEM(Dulbeco’s Modified Eagle’s Medium) , When the sample reached to confluence state, it was separated with 0,25% t rypsin and 0‘ 53mM ethylendiaminetetraacetic aCld Separated cells were centrifuged in a cell culturing flask at 1000rpm for 30, 60, 120 mmutes Then it was forced by 35g/cm' continuously, The obtained results that expression of mRNA using histological study and Reverse Transcription Polymerase Chain Reaction (RT-PCR) , expression of protein using Enzyme-Linked Immunosorbent Assay(ELISA) for this study, At 30minutes after cen trifuging, there were s pindl e shaped gingival fibroblasts with long processes parallel to other cells in the control group , However the cell density was simil ar to compared group, At 60minutes after centrifuging, spindle shaped human gingival fibroblast with relatively long process, less densely packed, At 120minutes after centrifuging, cell processes were lengthened 2-3 times‘ and cell density was lower, At 30-60 minutes after centrifuging, it was increased by 1,3-1,7 times in expressoin of collagen 1 mRNA as compared with comparison group, However, there was no change in elastin, TIMP-l, and MMP-1, At 120 minutes after centrifuging, The revealed collagen 1 mRNA was increased 3 times as compared with comparison group, It was increased 2 times in elastin , 12 times in TIMP-1 as compared with comparison group, However, there was no change in MMP-l. At 30-60 minutes after centrifuging, it was increased by 1.1 times in revealing of protein revealing in collagen 1, TIMP-1 But there was no cha nge in elastin, MMP-1 At 120 minutes after centrifuging, it was increased by 1,2 times in revealing collagen 1 protein, 11 times in elastin, 12 times in TIMP-l, but there was no change in MMP-l. ln conclu s ion, it increased in revelation of collagen 1 ,elastin and TIMP-1 by continous stimulus in human gi ngival fibroblast, But there was no change in revelation of MMP-l Therefore, th is type of pressu re is one of the components for healing of gingiva l fibroblast

It has been reported that light-emitting diodes(LED) can be used in the treatment of oral diseases. Although bio-stimulatory effects of LED irradiation such as promotion of wound healing have been well known, there are few reports about molecular mechanisms associated with wound healing by LED irradiation. The purpose of the present study was to investigate the expression pattern of various extracellular matrix(ECM) molecules in relation to wound healing after LED irradiation on primary human gingival fibroblasts(hGFs) in vitro. The source of light for irradiation was a continuous-wave LED emitting at a wavelength of 635 nm, and manufactured that energy density was 5 mW/cm2 on sample surfaces. The hGFs were irradiated for 1 hour at 37℃ in 5% CO2 humidified chamber. Experimental samples were acquired at 0 (right after irradiation), 24 and 48 hour after irradiation. To investigate the molecular mechanisms associated with wound healing, we examined the mRNA expression of 6 types of collagens, 7 types of matrix metalloproteinases(MMPs) and 4 types of tissue inhibition of metalloproteinases(TIMPs) after LED irradiation by RT-PCR. The mRNA expression of collagen 4, MMP-3, 9, and 16, and TIMP-3 was influenced by LED irradiation. Generally, the collagen expression of the irradiation group was slightly increased, particularly collagen 4 was significantly increased at 0 hour. The expression of MMP-3 was increased at 0 and 24 hours and MMP-16 was increased at 24 hours, respectively. The expression of MMP-9 was decreased at 0 hour and increased at 24 and 48 hours. The mRNA expression of TIMP-3 was significantly decreased at 24 and 48 hours after irradiation. These results suggest that the altered expression of ECM molecules after LED irradiation may contribute to the accelerated wound healing.

Treponema maltophilum, a Group IV oral spirochete, is associated with periodontitis and endodontic infections. In this study we analyzed the functional role of the major surface protein of this organism (MspA) in human gingival fibroblasts (HGFs). The full-length gene encoding MspA was cloned and expressed in Escherichia coli by using the expression vector pQE-30. The recombinant protein (rMspA) was purified by affinity chromatography with nickel-nitrilotriacetic acid agarose and possible contamination of E. coli endotoxin in rMspA was removed by using polymyxin B-agarose. rMspA significantly induced the expression of pro inflammatory cytokines like IL-6 and IL-8 and intercellular adhesion molecule (ICAM)-1 in HGFs, when analyzed by reverse transcription-PCR, flow cytometry, and enzyme-linked immunosorbent assay. Our results indicate that MspA of T. maltophilum may play an important role in amplifying the local immune response by upregulating the expression of proinflammatory cytokines and ICAM-1.