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Lidocaine-Induced Cell Growth of Human Gingival Fibroblasts. Role of Na+-K+-ATPase and PKC Activities  [PDF]
Emmanuel Quinteros Villarruel, Betina Orman, Enri Borda
Pharmacology & Pharmacy (PP) , 2014, DOI: 10.4236/pp.2014.58090

Background: Evidences have shown that local anaesthetics are clinically useful compounds that exert a pharmacological effect by blocking nerve impulse propagation and also it is able to provoke proliferation and cell growth. Aims: The aim of this study was to investigate the proliferation and cell growth capacity of lidocaine on human gingival fibroblast cells and the different signal pathways involved in its effect. Method: For this purpose in vitro cultures of human gingival fibroblasts were assayed and the effects of lidocaine on proliferation and cell DNA synthesis, Na+-K+-ATPase and PKC activities and K+ efflux were also evaluated. Results: Lidocaine stimulated in a concentration-dependent manner proliferation and cell growth of human gingival cells and the mechanism involve an increment in Na+-K+-ATPase and PKC activities, which led to an increase in K+ release. All of these effects were blocked by tetrodotoxin, ouabain and calphostin C. In addition, PMA (activator of PKC)

Primary cell culture from human oral tissue: gingival keratinocytes,gingival fibroblasts and periodontal ligament fibroblasts  [PDF]
Supreya Wanichpakorn,Ureporn Kedjarune-Laggat
Songklanakarin Journal of Science and Technology , 2010,
Abstract: Primary cell culture of human oral tissue has many applications for oral biology research. There are two techniques in primary culture, which includes the enzymatic and direct explant technique. The objectives of this study were (1) to isolate and investigate the difference in percentage the success in culturing three cell types from human oral tissue: gingival keratinocytes, gingival fibroblasts and periodontal ligament fibroblasts by using the direct explant technique; (2) to compare the effect of sex and age on the success of tissue culturing. Twenty seven tissue samples were obtained from healthy human gingival tissue, 19 female and 8 male patients aged 14-67 years (37.7±17.5). The tissue was cut into 1x1 mm pieces and placed on plastic culture plates containing Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 1% amphotericin B. For the keratinocytes culture, after the epithelial cells started to multiply around the gingival origin and the diameter was 2-5 mm., the fibroblasts were liminated by mechanical removal under inverted microscope to prevent fibroblast overgrowth and the medium was changed to keratinocyte-SFM (Gibco, BRL) supplemented with 5 μg/ml gentamycin. The results revealed that gingival fibroblast gave the highest success rate in culture (96.3%), followed by gingival keratinocytes (88.9%) and periodontal ligament fibroblasts (81.5%). There was no significant difference in the success rate of cultivation between younger and older individuals, as between sex of the subjects (p>0.05). The risk of failure in culture techniques is mainly caused by microbiological contamination from the tissue samples.
In vitro assessment of cytotoxicity of giomer on human gingival fibroblasts
R Pourabbas, S Farajnia, S Kimya, L Mohammadnejad, A Johnson, T Nejatian
African Journal of Biotechnology , 2009,
Abstract: Root coverage on restored root surfaces has been considered as a challenging issue. The evaluation of cytotoxic effects of restorative materials is a fundamental requirement for sustaining the cell attachment and the clinical success of root coverage. The aim of the present study was to compare the human gingival fibroblast cytotoxicity of the recently introduced giomer composite (GC) with resin ionomer (RI) restorative material. Discs (6×2 mm) of GC and RI restorative materials were prepared using sterile Teflon mold. Extracts from the materials were incubated to cell culture medium for 24, 48 and 72 h. Human gingival fibroblasts (HGF) were exposed to the extracts of the materials while the unincubated media served as the control group. The cytotoxicity of the materials were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In order to compare the mean values of the measured parameters a Kruskal-Walis test was carried out. MTT assay indicated that human gingival fibroblasts proliferated well in the presence of GC extract. The proliferation rate was higher in cells incubated with GC compared to RI extracts but the differences were not statistically significant (p= 0.09). This in vitro study indicated that GC is a non-toxic material for HGF. However, further studies are needed to assess the other biologic and clinical behavior of this material prior to it being considered as a potentially suitable restorative material to restore the carious root lesions candidated to root coverage procedures.
A simple and established method of tissue culture of human gingival fibroblasts for gingival augmentation.  [cached]
Jolanta Saczko,Marzena Dominiak,Julita Kulbacka,Agnieszka Chwi?kowska
Folia Histochemica et Cytobiologica , 2008, DOI: 10.5603/4451
Abstract: Recent advances in tissue engineering technology suggest its application in different medical fields, including periodontology. There are some reports of new non-enzymatic methods of isolating human gingival fibroblast for short-time cultivation in vitro to be used in autologous gingival augmentation. The aim of this study was to obtain a simple and established method of culturing human gingival fibroblasts. The authors developed a recurrent method that can be successfully used in autologous gingival augmentation.
Isolation and characterization of gingival fibroblasts positive for alkaline phosphatase in patients with chronic periodontitis and drug-induced gingival hyperplasia
Santos, Vagner Rodrigues;Gomes, Rafael Tomaz;Resende, Maurício;Almeida, Oslei Paes de;Colleta, Ricardo Della;
Revista Odonto Ciência , 2010, DOI: 10.1590/S1980-65232010000100011
Abstract: purpose: to investigate the expression of alkaline phosphatase (alp) activity in gingival fibroblasts from individuals with chronic periodontitis (cp) and drug-induced gingival hyperplasia (dgh) induced by diphenylhydantoin. methods: gingival fragments were obtained from 13 patients (8 women and 5 men, from 22 to 74 years of age), with 4 fragments from clinically normal gingiva (ng), 5 from biopsy of periodontal pockets with cp, and 4 from dgh induced by diphenylhydantoin. using an enzymatic digestion procedure, gingival cell suspensions containing alp-positive fibroblasts were prepared without affecting alp activity. cytochemistry and histochemistry analyses were performed. results: fibroblasts from ng presented low levels of alp when compared to cp and dgh, which showed elevated and intermediate levels of alp, respectively. little cell proliferation was observed for fibroblasts from cp and dgh as compared to ng. however, the quantity of cells recovered from the subcultures was similar to the quantity recovered from the initial cell culture for the three sources. conclusion: the expression of alp is increased in cp and dgh, and fibroblasts in cp and dgh show low proliferation. this suggests that periodontal inflammation and diphenylhydantoin may influence alp expression and human gingival fibroblast expansion. other studies are necessary to better assess the importance of alp in the development and progression of cp and dgh.
Activity of 25-Hydroxylase in Human Gingival Fibroblasts and Periodontal Ligament Cells  [PDF]
Kaining Liu, Huanxin Meng, Jianxia Hou
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0052053
Abstract: Background We previously demonstrated that 25-hydroxyvitamin D3 concentrations in gingival crevicular fluid are 300 times higher than those in the plasma of patients with aggressive periodontitis. Here we explored whether 25-hydroxyvitamin D3 can be synthesized by periodontal soft tissue cells. We also investigated which of the two main kinds of hydroxylases, CYP27A1 and CYP2R1, is the key 25-hydroxylase in periodontal soft tissue cells. Methodology/Principal Findings Primary cultures of human gingival fibroblasts and periodontal ligament cells from 5 individual donors were established. CYP27A1 mRNA, CYP2R1 mRNA and CYP27A1 protein were detected in human gingival fibroblasts and periodontal ligament cells, whereas CYP2R1 protein was not. After incubation with the 25-hydroxylase substrate vitamin D3, human gingival fibroblasts and periodontal ligament cells generated detectable 25-hydroxyvitamin D3 that resulted in the production of 1α,25-dihydroxyvitamin D3. Specific knockdown of CYP27A1 in human gingival fibroblasts and periodontal ligament cells using siRNA resulted in a significant reduction in both 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 production. Knockdown of CYP2R1 did not significantly influence 25-hydroxyvitamin D3 synthesis. Sodium butyrate did not influence significantly CYP27A1 mRNA expression; however, interleukin-1β and Porphyromonas gingivalis lipopolysaccharide strongly induced CYP27A1 mRNA expression in human gingival fibroblasts and periodontal ligament cells. Conclusions The activity of 25-hydroxylase was verified in human gingival fibroblasts and periodontal ligament cells, and CYP27A1 was identified as the key 25-hydroxylase in these cells.
Effect of Protein Kinase C delta (PKC-δ) Inhibition on the Transcriptome of Normal and Systemic Sclerosis Human Dermal Fibroblasts In Vitro  [PDF]
Peter J. Wermuth, Sankar Addya, Sergio A. Jimenez
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0027110
Abstract: Previous studies demonstrated that protein kinase C- δ (PKC-δ) inhibition with the selective inhibitor, rottlerin, resulted in potent downregulation of type I collagen expression and production in normal human dermal fibroblasts and abrogated the exaggerated type I collagen production and expression in fibroblasts cultured from affected skin from patients with the fibrosing disorder systemic sclerosis (SSc). To elucidate the mechanisms involved in the ability of PKC-δ to regulate collagen production in fibroblasts, we examined the effects of PKC-δ inhibition on the transcriptome of normal and SSc human dermal fibroblasts. Normal and SSc human dermal fibroblasts were incubated with rottlerin (5 μM), and their gene expression was analyzed by microarrays. Pathway analysis and gene ontology analysis of differentially expressed genes in each comparison were performed. Identification of significantly overrepresented transcriptional regulatory elements (TREs) was performed using the Promoter Analysis and Interaction Network Toolset (PAINT) program. PKC-δ activity was also inhibited using RNA interference (siRNA) and by treating fibroblasts with a specific PKC-δ inhibitory cell permeable peptide. Differential gene expression of 20 genes was confirmed using real time PCR. PKC-δ inhibition caused a profound change in the transcriptome of normal and SSc human dermal fibroblasts in vitro. Pathway and gene ontology analysis identified multiple cellular and organismal pathways affected by PKC-δ inhibition. Furthermore, both pathway and PAINT analyses indicated that the transcription factor NFκB played an important role in the transcriptome changes induced by PKC-δ inhibition. Multiple genes involved in the degradation of the extracellular matrix components were significantly reduced in SSc fibroblasts and their expression was increased by PKC-δ inhibition. These results indicate that isoform-specific inhibition of PKC-δ profibrotic effects may represent a novel therapeutic approach for SSc and other fibrotic diseases.
Effects of hyaluronan oligosaccharides on apoptosis ofhuman gingival fibroblasts  [PDF]
Yuki Tanne, Kotaro Tanimoto, Satoru Okuma, Ryo Kunimatsu, Naoto Hirose, Tomomi Mitsuyoshi, Kazuo Tanne
Open Journal of Stomatology (OJST) , 2013, DOI: 10.4236/ojst.2013.31004

An increased content of low molecular weight (LMW)- HA was found in human gingiva during the initial phase of periodontitis. A number of functions have been described for LMW-HA in inflammatory processes. It is also demonstrated that LMWHA induces apoptosis in chondrocytes and tumor cells, however, effects of LMWHA on the apoptosis of gingival fibroblasts (GFs) remain unclear. The purpose of this study was to investigate the effects of HA oligosaccharides (HAoligo) on the apoptosis of GFs.

Effects of bFGF on the Modulation of Apoptosis in Gingival Fibroblasts with Different Host Ages  [PDF]
Kotaro Tanimoto,Satoru Ohkuma,Yuki Tanne,Ryo Kunimatsu,Naoto Hirose,Tomomi Mitsuyoshi,Yuki Yoshimi,Shaoching Su,Kazuo Tanne
International Journal of Dentistry , 2013, DOI: 10.1155/2013/619580
Abstract: The purpose of this study was to investigate the effects of basic fibroblast growth factor (bFGF) treatment on the proliferation and apoptosis of cultured gingival fibroblasts (GFs). Human GFs were isolated from the palatal gingival tissues of 16 healthy volunteers ranging in the age from 9 to 35 years old. Cultured GFs were subjected to the analyses for cell proliferation by ELISA assay, gene expression by RT-PCR analysis, and apoptosis potency by caspase-3 assay. The cell proliferation activity and gene expression of type-I collagen and caspase-3 activity were enhanced significantly by the treatment with bFGF in cultured GFs. Furthermore, the activity of caspase-3 in cultured GFs from young subjects was significantly higher than that in GFs from adults. It is shown that bFGF significantly enhances the gene expression of type-I collagen in cultured fibroblasts from human gingival tissues. It also demonstrated that bFGF modulates the apoptosis of periodontal fibroblasts, and the effect is higher in young subjects, indicating a significant role of bFGF in the prevention of scar formation during wound healing. 1. Introduction Basic fibroblast growth factor (bFGF) is a multigene family member of structurally related peptide growth factors, and its function is mediated through high-affinity receptors [1]. It is well known that bFGF is a multifunctional cytokine to participate in the process of wound healing, cell proliferation, and apoptosis [2–4]. Wound healing can be divided into three consecutive, partly overlapping phases: inflammation, proliferation, and tissue remodeling [5]. The macrophage plays a pivotal role in the transition between wound inflammation and repair, since this cell both scavenges tissue debris and releases a plethora of biologically active substances that include growth factors like bFGF. During the remodeling phase, the number of blood vessels declines and apoptosis of fibroblasts results in scar tissue with a low cell density [6]. Apoptosis is a requisite event for maintaining kinetic homeostasis in continuously renewing tissues such as oral mucosa and skin, and it is suggested to play a crucial role in the repair of connective tissues. Nevertheless, apoptosis is often involved in pathogenetic pathways [7]. Regarding the mechanism of apoptosis induced by bFGF treatment, it has recently been demonstrated that bFGF plays an important role in apoptosis during development of the neural retina. The apoptosis of fibroblasts treated with bFGF was enhanced in both in vivo and in vitro experiments [8, 9]. In dentistry, bFGF was reported to
Gingival Fibroblasts Display Reduced Adhesion and Spreading on Extracellular Matrix: A Possible Basis for Scarless Tissue Repair?  [PDF]
Fen Guo, David E. Carter, Anuradha Mukhopadhyay, Andrew Leask
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0027097
Abstract: Unlike skin, oral gingiva do not scar in response to injury. The basis of this difference is likely to be revealed by comparing the responses of dermal and gingival fibroblasts to fibrogenic stimuli. Previously, we showed that, compared to dermal fibroblasts, gingival fibroblasts are less responsive to the potent pro-fibrotic cytokine TGFβ, due to a reduced production of endothelin-1 (ET-1). In this report, we show that, compared to dermal fibroblasts, human gingival fibroblasts show reduced expression of pro-adhesive mRNAs and proteins including integrins α2 and α4 and focal adhesion kinase (FAK). Consistent with these observations, gingival fibroblasts are less able to adhere to and spread on both fibronectin and type I collagen. Moreover, the enhanced production of ET-1 mRNA and protein in dermal fibroblasts is reduced by the FAK/src inhibitor PP2. Given our previous observations suggesting that fibrotic fibroblasts display elevated adhesive properties, our data suggest that scarring potential may be based, at least in part, on differences in adhesive properties among fibroblasts resident in connective tissue. Controlling adhesive properties may be of benefit in controlling scarring in response to tissue injury.
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