Cultures Of Gingival Fibroblasts Research Articles

Two Functions of Lysyl Oxidases: Extracellular Matrix Maturation and Cell Proliferation

Lysyl oxidases are enzymes which catalyze the final enzymatic modification of ε‐ amino groups of lysine residues of elastin, and lysine and hydroxylysine residues of collagens, to form peptidyl‐aldehydes required for biosynthetic cross‐linking and extracellular matrix maturation and function. This family includes lysyl oxidase (LOX) and lysyl oxidase‐like 1 – 4 (LOXL1 – LOXL4). CCN2 contributes to fibrosis development in gingival overgrowth caused by the anti‐seizure drug phenytoin, though the mechanism CCN2 is unclear. Here we investigate the hypothesis that CCN2 could up‐regulate LOXL2 to promote gingival fibroblast insoluble collagen deposition.CCN2 up‐regulated LOXL2 protein levels in primary human gingival fibroblast cultures. shRNA‐mediated knockdown of LOXL2 blocked collagen accumulation in CCN2‐treated and non‐treated primary human gingival fibroblast cultures determined by Sirius Red staining. DNA in cell layers was also strongly diminished in cell layers. Cell proliferation was decreased in LOXL2 shRNA transduced cells, while apoptosis was not affected. Active LOXL2 enzyme addition promoted both increased proliferation and cell layer collagen accumulation.LOXL2 has two important functions in the development of gingival fibrosis: its known role in promoting collagen maturation, and the new role of promoting cell proliferation. The mechanism of promotion of proliferation of gingival fibroblasts by LOXL2 is under investigation.Supported by R01 DE11004.

Cytotoxic and genotoxic effects on gingival fibroblasts from static magnetic fields produced by dental magnetic attachments.

To investigate cytotoxic and genotoxic effects of static magnetic field (SMF) produced by dental magnetic attachments on human gingival fibroblasts in vitro. Magnetic attachments have numerous roles in dental prosthesis fixation, but few reports evaluate possible biological effects of static magnetic field (SMF) on human gingival tissues, particular genotoxic effects. The Dyna (500-gr breakaway force) and Steco (173-gr breakaway force) dental magnetic attachments were embedded into autopolymerising acrylic resin in four different configurations each, including single and double magnets. Gingival biopsy was performed on 28 individuals during third molar extraction, and each sample was divided into two pieces for culture under SMF exposure or as a control. In total, seven test and seven control gingival fibroblast cultures were performed for each group resulting in 56 gingival fibroblast cultures. The test culture flasks were placed atop the magnet-embedded resin blocks. After cultures were terminated, mitotic index (MI) and micronucleus (MN) rates were analysed at a p=0.05 significance level by Wilcoxon's test; intergroup differences were analysed with a Kruskal-Wallis test. There was no significant difference in intragroup or intergroup MI rates. The double Dyna (p=0.023) and double Steco (p=0.016) groups had statistically significant intragroup differences in the MN rates. There were no statistically significant differences in MN rates in intergroup analyses. In particular, higher magnetic fields from dental magnetic attachments might be toxic genetically to human gingival fibroblasts. However, there is need for further investigations from different aspects to detect any genotoxicity.

Biological evaluation of enamel sealants in an organotypic model of the human gingiva

ObjectivesVarious sealant materials have been suggested to decrease decalcification during orthodontic treatment. However, only a few in vitro studies on the cytotoxicity of resinous pit and fissure sealants have been published, and to the best of our knowledge no similar studies are available for the enamel sealants used in orthodontics. Therefore, we aimed to characterize the possible adverse effects of enamel sealants, especially on the gingival epithelium. MethodsOrganotypic cultures of the human gingival mucosa were used to assess the possible impact of six enamel sealants. Differentiation and apoptosis were determined by immunofluorescent staining. The pro-inflammatory cytokines IL-1β and IL-6 were quantified by ELISA. Cytotoxicity was measured using MTS assays in monolayer cultures of human gingival fibroblasts. Leaching of monomers from enamel sealants was quantified using HPLC. ResultsThe differentiation of the organotypic gingival mucosa remained unaffected. All under-cured and several standard-cured sealants (Light Bond™ Sealant, Light Bond™ Filled Sealant, and L.E.D. Pro Seal®) significantly induced apoptosis in the organotypic model. Light Bond™ Sealant, Light Bond™ Filled Sealant, and L.E.D. Pro Seal® caused a significant induction of pro-inflammatory cytokines. Reducing curing time had an influence on cytotoxicity in monolayer cultures of primary human oral cells. All resin-based sealants leached monomers. SignificanceEnamel sealants might exert adverse effects on the gingival epithelium. Due to the vicinity of the enamel sealant to the gingival epithelium, and the large surface area of applied sealants, these materials should be carefully applied and sufficiently cured.

Cell culture of human gingival fibroblasts, oral cancer cells and mesothelioma cells with serum-free media, STK1 and STK2.

The majority of cells are cultured with Dulbecco's modified Eagle's medium (DMEM) or RPMI supplemented with fetal bovine serum (FBS), which contains numerous factors, including cytokines, nutrients and unknown growth factors. These factors may affect cell growth, apoptosis and differentiation. The serum-free medium, STK2, has been previously reported as suitable for the cell culture of human mesenchymal stem cells. However, how STK1 or STK2 affect the cell proliferation of normal and cancer cells remains unknown. The present study examined the growth of the human gingival fibroblast (HGF-1) cell-line and the HSC-3, CA9-22 and MSTO cancer cell-lines, cultured with STK1 and STK2. STK1 increased the cell proliferation of HGF-1 compared to DMEM by assessment with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium (MTS) assay, whereas STK1 and STK2 markedly inhibited the cell proliferation of HSC-3 and MSTO. The cell proliferation rate of CA9-22 cultured with STK1 or STK2 for 96 h was ~2-fold higher than the rate for 24 h culture. The shape of the HSC-3 cells was also found to have changed to round when cultured with STK2. These results indicate that STK1 increased the cell proliferation of HGF-1 compared to DMEM, whereas the proliferation of HSC-3 and MSTO was inhibited by STK1 and STK2. Thus, STK1 and STK2 had different affects on the cell growth of HGF-1, CA9-22, HSC-3 and MSTO.

Effect of novobiocin on the viability of human gingival fibroblasts (HGF-1).

BackgroundNovobiocin is a coumarin antibiotic, which affects also eukaryotic cells inhibiting activity of Heat shock protein 90 (Hsp90). The Hsp90 represents a molecular chaperone critical for stabilization and activation of many proteins, particularly oncoproteins that drive cancer progression. Currently, Hsp90 inhibitors focus a significant attention since they form a potentially new class of drugs in therapy of cancer. However, in the process of tumorigenesis a significant role is played also by the microenvironment of the tumour, and, in particular, by cancer-associated fibroblasts (CAFs). This study aimed at examination of the effect played by novobiocin on viability of human gingival fibroblasts (HGF-1).MethodsThe studies were conducted using 24 h cultures of human gingival fibroblasts – HGF-1 (CRL-2014) in Chamber Slides, in presence of 0.1, 0.5, 1.0, 2.5 or 5.0 mM novobiocin. Cell viability was evaluated using fluorescence test, ATP assay and LDH release.ResultsViability of HGF-1 was drastically reduced after 5 hour treatment with novobiocin in concentrations of 1 mM or higher. In turn, the percentage of LDH-releasing cells after 5 h did not differ from control value although it significantly increased after 10 h incubation with 1 mM and continued to increase till the 20th hour.ConclusionsThe obtained data indicate that novobiocin may induce death of human gingival fibroblasts. Therefore, application of the Hsp90 inhibitor in neoplastic therapy seems controversial: on one hand novobiocin reduces tumour-associated CAFs but, on the other, it may induce a significant destruction of periodontium.

Defective Wound-healing in Aging Gingival Tissue

Aging may negatively affect gingival wound-healing. However, little is known about the mechanisms underlying this phenomenon. The present study examined the cellular responses associated with gingival wound-healing in aging. Primary cultures of human gingival fibroblasts were obtained from healthy young and aged donors for the analysis of cell proliferation, cell invasion, myofibroblastic differentiation, and collagen gel remodeling. Serum from young and old rats was used to stimulate cell migration. Gingival repair was evaluated in Sprague-Dawley rats of different ages. Data were analyzed by the Mann-Whitney and Kruskal-Wallis tests, with a p value of .05. Fibroblasts from aged donors showed a significant decrease in cell proliferation, migration, Rac activation, and collagen remodeling when compared with young fibroblasts. Serum from young rats induced higher cell migration when compared with serum from old rats. After TGF-beta1 stimulation, both young and old fibroblasts demonstrated increased levels of alpha-SMA. However, alpha-SMA was incorporated into actin stress fibers in young but not in old fibroblasts. After 7 days of repair, a significant delay in gingival wound-healing was observed in old rats. The present study suggests that cell migration, myofibroblastic differentiation, collagen gel remodeling, and proliferation are decreased in aged fibroblasts. In addition, altered cell migration in wound-healing may be attributable not only to cellular defects but also to changes in serum factors associated with the senescence process.

Expression of cathepsin K in periodontitis and in gingival fibroblasts

To study non-osteoclastic sources of cathepsin K in periodontitis. Tissue samples were obtained from 10 otherwise healthy periodontitis pati-ents during routine periodontal flap operations and 10 systemically and periodontally healthy individuals who underwent extraction operations for retained third molars. Methods used were immunohistochemistry, image analysis, immunofluorescence double-staining, gingival fibroblast culture, tumour necrosis factor-α (TNF-α) stimulation and Western blotting. Macrophage-like cells, fibroblast-like cells, vascular endothelial cells and gingival epithelial cells were more intensively stained for cathepsin K and also more frequent in periodontitis than in controls (665±104 vs 258±40cellsmm(-2) , P<0.01). Some cathepsin K(+) cells in periodontal tissues were CD68(+) , but some were CD68(-) and probably fibroblasts. Indeed, in gingival fibroblast culture, resting fibroblasts released cathepsin K, more 43kD procathepsin K than 29kD active cathepsin K. TNF-α increased the release of the activated cathepsin K 4- to 5-fold. Results suggest that GCF-cathepsin K is not only osteoclast-derived, but in periodontitis, also other cells contribute to it. GCF-cathepsin K, perhaps together with intracellular, lysosomal collagenolytically active cathepsin K in fibroblasts, macrophages and gingival epithelial cells, can contribute to the loss of attachment and destruction of the periodontal ligament.

Differential expression of Toll‐like receptor 4 in healthy and diseased human gingiva

Lipopolysaccharide (LPS)-mediated signaling in host cells involves Toll-like receptor 4 (TLR4) accessory molecules, including LPS-binding protein (LBP), cluster of differentiation 14 (CD14) and lymphocyte antigen 96 (MD-2). However, expression of these innate defense molecules in various compartments of the human periodontium is unclear. The aim of this study was to investigate the expression profile of TLR4 in human gingiva. Human gingival biopsies were collected from healthy gingival or chronic periodontitis tissue. Primary gingival keratinocytes and fibroblasts were cultured. Immunohistochemical analysis for TLR4 was performed. Transcripts of TLR4, MD-2, CD14 and LBP, and their protein products, were examined using RT-PCR, immunoprecipitation and immunoblotting. The interactions between these molecules in keratinocytes and fibroblasts were investigated by co-immunoprecipitation. TLR4 immunoreactivity was found in healthy gingival epithelium and periodontitis tissue, and appeared to be lower in junctional epithelium (p≤0.01). Fibroblasts and inflammatory cells stained more strongly for TLR4 in diseased periodontal tissues (p<0.001). Three TLR4 splicing variants, two MD-2 splicing variants and one CD14 mRNA were expressed by gingival keratinocytes and fibroblasts. Expression of TLR4, CD14 and MD-2 proteins was detected in keratinocytes and fibroblasts in vitro. TLR4 protein from gingival keratinocytes and fibroblasts could be co-immunoprecipitated with CD14 or MD-2, suggesting an association between the related molecules in vivo. LBP transcript was detected in gingival biopsies, but not in primary cultures of gingival keratinocytes or fibroblasts. TLR4, CD14 and MD-2, but not LBP, are expressed in human gingival keratinocytes and fibroblasts. The TLR4 expression level in the junctional epithelium appeared to be lowest within the periodontal epithelial barrier.

Effect of Electron Beam Irradiation on Resin– Based Root Canal Sealer –AH Plus: A Cytotoxic Evaluation

Aims: To evaluate and compare the cytotoxicity of a resin-based root canal sealer, AH Plus, before and after electron beam irradiation and to assess the effect of three different doses of electron beam radiation (1 KGy, 3 KGy and 5 KGy) on cytotoxicity of the sealer. Place and Duration of Study: Department of Conservative Dentistry and Endodontics, A.B. Shetty Memorial Institute of Dental Sciences, Nitte University, Deralakatte, Mangalore, India and Microtron Centre; Department of Physics Mangalore University; Mangalore, India between July 2012 and November 2012. Methodology: Gingival fibroblasts were cultured in Dulbecco’s Modified Eagle’s Medium Research Article Annual Research & Review in Biology, 4(1): 163-173, 2014 164 (DMEM) and sub confluent monolayers of cells were obtained. The study included two categoriesIrradiated and Non-irradiated. Both of them were further divided into four groups of paste A, paste B, Mixed and Set sealer of AH plus. Samples in irradiated category were further divided into 3 subgroups based on electron beam irradiation dose-1, 3 and 5 KGy. Sample elutes were prepared using standard extraction procedure. The elutes of all sealers from both the categories were added to the human gingival fibroblast cultures and their cytotoxicity was assessed by MTT assay. The results were tabulated and subjected to statistical analysis using One–way ANOVA (Analysis of variance) test and Tukey’s HSD Test. Results: Cell viability was minimum in Paste M irradiated with 5 KGy irradiation dose and maximum in non-irradiated SET AH Plus sealer. The results of the present study showed positive effect of irradiation in the epoxide paste of AH Plus sealer. The positive effect here is mainly based on experimental data of percentage cell viability and not on statistical significance. Multiple comparisons of different doses of electron beam irradiation on percentage of cell viability in all the samples showed a highly statistically significant difference at 3 KGy of electron beam radiation (P value = 0.008). Conclusion: Further research should to be performed with higher doses of electron beam radiation on epoxide paste in order to notice further improvement in physical as well as biological properties in order to generate an upgraded version of the present dental materials.

Lidocaine-Induced Cell Growth of Human Gingival Fibroblasts. Role of Na&amp;lt;sup&amp;gt;+&amp;lt;/sup&amp;gt;-K&amp;lt;sup&amp;gt;+&amp;lt;/sup&amp;gt;-ATPase and PKC Activities

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) increased per se the DNA synthesis of human gingival fibroblast cells. Conclusions: This work demonstrates that lidocaine increase human gingival fibroblasts DNA synthesis and proliferation through an activation of PKC pathway accompanied by the stimulation of Na+-K+-ATPase activity with an increase in K+ efflux. These results contribute to showing another action of lidocaine different to its general use as a drug that relieves odontologic pain or acts as an anti-arrithmogenic agent.

Ultrastructural changes in human gingival fibroblasts after exposure to 2-hydroxy-ethyl methacrylate.

Polymerized resin-based materials are successfully utilized in medical applications. One draw- back is the release of monomers from the matrix due to an incomplete polymerization or degradation processes. Released monomers can diffuse in the systemic circulation and induceadverse effects to biological tissues. Although there are many hypotheses about the induction of cell death by resin monomers, the underlying mechanisms are still under discussion. The aim of the study was to investigate the morphological modifications in human gingival fibroblasts exposed to 2-hydroxy-ethyl methacrylate (HEMA) to better elucidate the mechanism of cell death induced by resin monomers. Primary cultures of gingival fibroblasts were exposed to 3mM HEMA for 24 h, 72 h, 96 h. Morphological investigations were performed by scanning and transmission electron microscopy, while western blot for caspase-3 was carried out to ver- ify apoptosis. Electron microscopy images showed deep changes in the cell surface and cyto- plasm after 72 h and 96 h of HEMA treatment. Autophagic vesicles were easily observed just after 24 h. Cleaved caspase-3 was detected after 72 h of treatment. These findings suggest that resin based materials induced cell death by the cooperation of apoptosis and autophagy mecha- nisms. The understanding of these mechanisms will lead to the development of smart biomate- rials without or with low adverse effects.

Cytotoxic Effects of Zoledronic Acid on Human Epithelial Cells and Gingival Fibroblasts

Bisphosphonate-induced osteonecrosis has been related to the cytotoxicity of these drugs on oral mucosa cells. A previous study showed that 5 µM of zoledronic acid (ZA), a nitrogen-containing bisphosphonate, is the highest concentration of this drug found in the oral cavity of patients under treatment. Therefore, in order to simulate an osteonecrosis clinical condition, the aim of this study was to evaluate the highest concentration of ZA applied on human epithelial cells (HaCaT) and gingival fibroblasts. For this purpose, cells (3 × 10(4) cells/cm2) were seeded in wells for 48 h using complete culture medium (cDMEM). After 48 h incubation, the cDMEM was replaced by fresh serum-free culture medium (DMEM-FBS) in which the cells were maintained for additional 24 h. Then, 5 µM ZA were added to the DMEM-FBS and the cells incubated in contact with the drug for 48 h. After this period, the number of viable cells (trypan blue), cell viability (MTT assay), total protein (TP) production and cell morphology (SEM analysis) were assessed. Data were analyzed statistically by Mann-Whitney, ANOVA and Tukey's test (α=0.05). ZA caused a significant reduction in the number of viable cells and decreased the metabolic activity of both cell lines. However, decrease of TP production occurred only in the epithelial cell cultures. Morphological alterations were observed in both cell types treated with ZA. In conclusion, ZA (5 µM) was cytotoxic to human epithelial cells and gingival fibroblast cultures, which could be associated, clinically, with the development of bisphosphonate-induced osteonecrosis.

Molecularly imprinted cryogels for human interferon‐alpha purification from human gingival fibroblast culture

Interferons are important proteins for the immune system because of their antiviral, anti-proliferating and immunomodulatory activities. Therapeutic value of these proteins against certain types of tumors caused interest and investigations aimed to obtain highly purified interferons. Molecular imprinting is an efficient method for purification with high selectivity, specificity and good reproducibility. In this study, we utilized advantages of molecular imprinting technique for the purification of interferon from human gingival fibroblast culture. For this purpose, interferon α-2b imprinted poly(hydroxyethyl methacrylate) cryogel (hIFN-α-MIP) was prepared. Optimum adsorption conditions were determined, and maximum adsorption capacity of hIFN-α-MIP cryogel was found as 254.8 × 10(4) IU/g from aqueous solution. All interferon measurements are expressed as International Unit (IU), which is a unit measurement used to quantify biologically active substances like interferon based on their biological activity or effect. Selectivity experiments were performed using competitive proteins and repeated adsorption-desorption studies showed that the adsorption capacity maintained almost at a constant value after ten cycles. For the purification of interferon from human gingival fibroblast culture, fast protein liquid chromatography was used and the specific activity of the purified interferon α-2b on HeLa cell line was found between the values 3.45 × 10(8) IU/mg and 3.75 × 10(8) IU/mg. The results are promising, and the molecular imprinting technique is effective for the purification of interferon α-2b.

Attachment, proliferation and collagen type I mRNA expression of human gingival fibroblasts on different biodegradable membranes

The purpose of this study was to investigate adhesion, proliferation and type I collagen (COL I) mRNA expression of gingival fibroblasts on different membranes used in periodontal applications. Collagen (C), acellular dermal matrix (ADM) and polylactic acid; polyglycolic acid; lactide/glycolide copolymer (PLGA) biodegradable membranes were combined with gingival fibroblasts in culture and incubated for 48 h. Cell adhesion was examined with scanning electron and confocal microscopy. MTT assay was used to measure proliferation. COL I mRNA expression was assessed using quantitative-polymerase chain reaction (QPCR). The PLGA group exhibited the lowest cell survival on day 5 and 10, and lowest cell proliferation on days 5, 10 and 14. While cell proliferation was similar in C and ADM groups, the C membrane showed a slightly greater increase in viable cells to day 10. Confocal and scanning electron microscopy confirmed the results of proliferation and MTT assays. The highest COL I mRNA expression was noted in the PLGA membrane group when compared to the C (p < 0.01) and ADM (p < 0.05) membrane groups. These data revealed that adherence and proliferation of primary gingival fibroblasts on collagen-based C and ADM membranes is better than that seen with PLGA membranes, and thus may be preferable in the treatment of gingival recession defects.

Human gingival fibroblast functions are stimulated by oxidized nano-structured titanium surfaces

ObjectivesThe aim of this study was to analyze the features of an oxidized titanium implant surface and to evaluate its effects on the response of human gingival fibroblasts. Methods10mm×10mm×1mm turned (control) and oxidized (test) titanium samples (P.H.I. s.r.l., Italy) were examined by scanning electron microscopy and atomic force microscopy and characterized by height, spatial and hybrid roughness parameters. Primary cultures of human gingival fibroblasts were seeded on titanium samples, and cell morphology, adhesion, proliferation and extracellular matrix deposition, in terms of type I collagen synthesis, were evaluated. ResultsControl and test surfaces appeared considerably different at the microscopic analyses: turned samples were grooved, whereas oxidized surfaces showed a more complex micro- and nano-scaled texture, as evidenced by roughness parameters. Cell adhesion and proliferation rate, as well as collagen synthesis, were greater on oxidized vs turned surfaces. ConclusionsAlthough both control and test samples were in the range of average roughness proper of smooth surfaces, they exhibited significantly different topographic properties in terms of height and, mostly, hybrid parameters. Furthermore, oxidized surfaces enhanced human gingival fibroblast adhesion, proliferation and extracellular matrix deposition, and this could be due to the different structure at micro- and nano-scale levels. Clinical significanceOxidized nanostructured titanium surfaces could have a significant clinical utilization in virtue of their affinity for soft tissue attachment at the implant neck and/or at the transmucosal portion of the prosthetic abutment.

Tumor Necrosis Factor‐α Inhibits Transforming Growth Factor‐β–Stimulated Myofibroblastic Differentiation and Extracellular Matrix Production in Human Gingival Fibroblasts

Fibroblasts play a critical role during wound healing and chronic inflammation through the synthesis and assembly of extracellular matrix (ECM) molecules. These responses may be modulated by soluble cytokines and growth factors present in tissues. In the present study, we evaluate whether transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) modulate myofibroblastic differentiation and the production of ECM components. Primary cultures of human gingival fibroblasts (HGFs) were stimulated with recombinant TGF-β1 and TNF-α. Protein levels of α-smooth muscle actin (α-SMA), type I collagen, heat shock protein-47 (HSP-47), fibronectin (FN), ED-A-FN, and periostin and activation of the Smad pathway were evaluated through Western blot analysis. α-SMA and actin fibers were identified by immunofluorescence. TGF-β1, TNF-α, and α-SMA were identified by immunohistochemistry in biopsies of inflamed human gingival tissues. TGF-β1 activity was evaluated using a plasminogen activator inhibitor-1 (PAI-1) reporter transfected in HGFs. TGF-β1 stimulated the differentiation of myofibroblasts as evidenced by strong expression of α-SMA and ED-A-FN. Moreover, TGF-β1 induced the production of type I collagen, HSP-47, FN, and periostin. Costimulation with TNF-α and TGF-β1 significantly reduced the expression of all the above-mentioned proteins. TNF-α also inhibited the activation of the Smad2/3 pathway and the activity of the PAI-1 reporter. TNF-α inhibits several cell responses induced by TGF-β1, including the differentiation of myofibroblasts, the activation of the Smad signaling pathway, and the production of key molecules involved in tissue repair, such as type I collagen, FN, and periostin. The interaction between cytokines may explain the delayed tissue repair observed in chronic inflammation of gingival tissues.

Chitosan and platelet‐derived growth factor synergistically stimulate cell proliferation in gingival fibroblasts

Chitosan is a naturally derived polymer that may be applied in periodontal therapy for tissue-reconstruction purposes. Previous studies have shown that chitosan may stimulate tissue healing. However, reports exploring the cellular responses stimulated by chitosan are lacking. In the present study we analyzed whether chitosan may promote cell proliferation in primary cultures of human gingival fibroblasts. Chitosan particles were generated, and their size, zeta potential and morphology were characterized using transmission and scanning electron microscopy and zetasizer analysis. The biocompatibility of chitosan particles was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell-viability assay and by detecting the release of lactate dehydrogenase into the cell-culture medium. The total number of cells was estimated by staining with crystal violet followed by measurement of the absorbance at 560nm on a microplate reader. Cell proliferation was studied by detecting proliferating cell nuclear antigen protein levels, immunofluorescence for Ki67 and incorporation of 5'-bromo-2'-deoxyuridine. The sizes of the chitosan particles generated were in the micrometer and nanometer ranges. Cell viability was increased in the presence of chitosan. Moreover, the combination of chitosan and platelet-derived growth factor (PDGF-BB) potently stimulated cell viability, cell proliferation and activation of the ERK1/2 pathway involved in cell proliferation. The present study shows that chitosan is well tolerated by gingival fibroblasts and is able to stimulate cell proliferation through the ERK1/2 signaling pathway. A synergistic response between chitosan and growth factors (such as PDGF-BB) may stimulate cell proliferation in gingival fibroblasts exposed to this biomaterial.

Anti-Inflammatory Activity of Selected Edible Herbs and Spices on Cultured Human Gingival Fibroblasts

The aims of the study were to determine the in vitro cytotoxicity and anti-inflammatory activities of common edible herbs and spices on cultured human gingival fibroblasts (HGFs). Piper betle L. (betel leaf), P.sarmentosum Roxb. (wild betel / kadok leaf), P.nigrum L. (black pepper seed), Eugenia caryophyllata L. (clove bud) and Cinnamomum zeylanicum Blume (cinnamon bark). Essential oils were extracted using steam distillation technique and analysed using gas chromatography (GC) and gas chromatography - mass spectrometry (GC-MS). The HGFs were exposed to essential oils at 5 - 0.04 µg/mL in less than 1% dimethylsulfoxide and the number of viable cells was counted to assess cytotoxicity effect. Anti-inflammatory action was determined via the inhibitory action of Interleukin-6 (IL-6), a major pro- inflammatory cytokine in the periodontal tissue inflammation. Treatment of fibroblasts with essential oils resulted in > 70% cell viability. The oils from black pepper seed, clove bud and cinnamon bark showed dose-dependent inhibitory action on IL-6 on cultured bacterial Lipopolysaccharide (LPS)-induced human gingival fibroblasts. Of all the oils, cinnamon bark oil showed the most prominent action comparable to acetylsalicylic acid. Conclusion: Essential oils of selected herbs and spices retained compatibility with gingival fibroblasts in culture and showed inhibitory activity on IL-6 released by LPS-induced HGFs. These findings suggest therapeutic potential for application of assay in the management of periodontal disease.

Perfused culture of gingival fibroblasts in a degradable/polar/hydrophobic/ionic polyurethane (D-PHI) scaffold leads to enhanced proliferation and metabolic activity

Periodontal diseases cause the breakdown of the tooth-supporting gingival tissue. In treatments aimed at gingival tissue regeneration, tissue engineering is preferred over the common treatments such as scaling. Perfused (dynamic) culture has been shown to increase cell growth in tissue-engineered scaffolds. Since gingival tissues are highly vascularized, it was desired to investigate the influence of perfusion on the function of human gingival fibroblasts (HGF) when cultured in a degradable/polar/hydrophobic/ionic polyurethane scaffold during the early culture phase (4weeks) of engineering gingival tissues. It was observed that the growth of HGF was continuous over 28days in dynamic culture (3-fold increase, p<0.05), while it was reduced after 14days in static culture (i.e. no flow condition). Cell metabolic activity, as measured by a WST-1 assay, and total protein production show that HGF were in different metabolic states in the dynamic vs. static cultures. Observations from scanning electron microscopy and type I collagen (Col I) production measured by Western blotting suggest that medium perfusion significantly promoted collagen production in HGF after the first 4weeks of culture (p<0.05). The different proliferative and metabolic states for HGF in the perfused scaffolds suggest a different cell phenotype which may favour tissue regeneration.

Screening and Scoring of Antimicrobial and Biological Activities of Italian Vulnerary Plants against Major Oral Pathogenic Bacteria

This study aims to evaluate the activity of Italian vulnerary plants against the most important oral pathogenic bacteria. This estimate was accomplished through a fivefold process: (a) a review of ethnobotanical and microbiological data concerning the Italian vulnerary plants; (b) the development of a scoring system to rank the plants; (c) the comparative assessment of microbiological properties; (d) the assessment of potential cytotoxic effects on keratinocyte-like cells and gingival fibroblasts in culture by XTT cell viability assay; (e) clinical evaluation of the most suitable plant extract as antibacterial agent in a home-made mouthwash. The study assays hexane (H), ethanol (E), and water (W) extracts from 72 plants. The agar diffusion method was used to evaluate the activity against Streptococcus mutans, Streptococcus sobrinus, Lactobacillus casei, and Actinomyces viscosus. Twenty-two plants showed appreciable activity. The extracts showing the strongest antibacterial power were those from Cotinus coggygria Scop., Equisetum hyemale L., Helichrysum litoreum Guss, Juniperus communis L., and Phyllitis scolopendrium (L.) Newman subsp. scolopendrium. The potential cytotoxic effect of these extracts was assessed. On the basis of these observations, a mouth-rinse containing the ethanolic extract of H. litoreum has been tested in vivo, resulting in reduction of the salivary concentration of S. mutans.