Primary Human Gingival Fibroblasts Research Articles

Mechanical properties and cell compatibility of a chitosan-graphene oxide guided bone regeneration composite membrane

Objective: To explore the feasibility of graphene oxide (GO)-chitosan (CS) guided bone regeneration composite membrane being used as a new guided bone regeneration (GBR) membrane by testing its tensile strength and its effect on the proliferation of human gingival fibroblasts (HGF). Methods: The CS solution and GO solution were mixed in following ratios, ultrasonically crushed and dispersed, and formed into membranes by self-evaporation. Composite membrane with 0.5%, 1.0%, 2.0%, 4.0%, 6.0%, 8.0% GO in CS was prepared. The tensile strength of the composite membranes was tested by mechanical universal testing machine (n=6). The microstructure of the composite membrane with the best tensile strength was observed by scanning electron microscopy and transmission electron microscopy. X-ray diffraction was used to characterize the ingredients of the material. A group of samples was prepared again with the proportion of the highest tensile strength composite membrane, and were immersed in a sodium hydroxide solution for acid removal. The tensile strength of the new group of samples was tested. The newly extracted impacted teeth were collected from the Department of Oral and Maxillofacial Surgery, Stomatologic Hospital and College, Anhui Medical University, and the gingival tissues remained on the teeth were taken for primary HGF and were cultured to P2 generation, which were identified by immunocytochemical staining methods. Cell counting kit-8 (CCK-8) was applied to detect cell counts in the blank control group, the pure CS membrane group, and the composite membrane group with the highest tensile strength (n=5) and all groups co-cultured with the HGF for 24 h and 48 h. Results: After adding GO, the cross-section of the composite membrane was in an ordered layer structure. X-ray diffraction analysis showed that GO could be found in the composite membrane. The tensile strength increased with the increase of GO ratio. When the mass fraction of GO in CS was 4.0%, the tensile strength reached (134.8±7.3) MPa and the tensile strength reached (144.6±8.1) MPa after deacidification of the composite membrane. The CCK-8 test of HGF showed that there was no significant difference in absorbance between the pure CS membrane group and the GO/CS composite membrane group when they were compared with the blank control group (P>0.05). Conclusions: When the mass fraction of GO in CS is 4.0%, the composite membrane has the best tensile strength. The composite membrane showes good cytocompatibility, which lays a foundation for further in vivo experiments and the development of a new generation of GBR membrane.

Investigation of foreign materials in gingival lesions: a clinicopathologic, energy-dispersive microanalysis of the lesions and in vitro confirmation of pro-inflammatory effects of the foreign materials

This study aimed to evaluate the clinical and histopathologic features of gingival lesions containing foreign material (GLFMs). In parallel, the composition of the foreign material and its effects in primary human gingival fibroblasts (HGFs) were investigated. Eighty-six GLFMs were retrieved from an oral pathology biopsy service. Clinical and microscopic data were analyzed, and the composition of the particles was identified by using energy-dispersive X-ray spectroscopy (EDX). Furthermore, HGFs were stimulated with silica (SiO2) microparticles to investigate the production of collagen type 1 (COL-1), matrix metalloproteinase 2 (MMP2), and inflammatory cytokines. GLFMs were most commonly found in women (60.5%) and most frequently described as white plaques. Histopathologic examination identified verrucous hyperplasia in 59% and epithelial dysplasia in 28% of the cases. EDX microanalysis revealed that Si (94%) was the most frequently detected foreign element. SiO2 microparticles induced higher COL-1 expression; higher levels of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-8, and transforming growth factor-β, and increased MMP-2 activity in HGFs. There was a strong association between the presence of foreign material in the gingiva and white verrucous clinical lesions. In addition, the most common element in the foreign material was Si, and our in vitro findings demonstrate the importance of silica-mediated effects on gingival fibroblasts.

In vitro cytotoxicity of different thermoplastic materials for clear aligners.

To investigate the in vitro cytotoxicity of different thermoplastic materials for clear aligners on human primary gingival fibroblasts (HGFs). Four materials for clear aligners were considered in this study: Duran (Scheu-Dental GmbH, Iserlohn, Germany), Biolon (Dreve Dentamid GmbH, Unna, Germany), Zendura (Bay Materials LLC, Fremont, CA, USA), and SmartTrack (Align Technology, San Jose, CA, USA). Three out of four materials (Duran, Biolon, Zendura) were assessed as thermoformed and nonthermoformed, whereas the SmartTrack was assessed only as thermoformed. The samples were placed at 37°C in airtight test tubes containing Dulbecco's Modified Eagle's Medium (DMEM; 0.1 mg/mL) for 14 days. The cell viability of HGFs cultured with this medium was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data were analyzed by means of one-way and two-way analysis of variance and post hoc tests (α = 0.05). Each material exhibited a slight cytotoxic effect after 14 days. The highest cytotoxicity level on HGFs was achieved by Biolon (64.6% ± 3.3 of cell viability), followed by Zendura (74.4% ± 2.3 of cell viability), SmartTrack (78.8% ± 6.3 of cell viability), and finally Duran (84.6% ± 4 of cell viability), which was the least cytotoxic. In the comparison between nonthermoformed and thermoformed materials for Duran, Biolon, and Zendura, the thermoformed materials showed the highest level of cytotoxicity (P < .001). Under the experimental conditions of this study, all the materials for clear aligners presented a slight cytotoxicity. Biolon was the most cytotoxic and the thermoforming process increased the cytotoxicity of the materials.

Characterization of different biodegradable scaffolds in tissue engineering.

The aim of the present study was to compare the characteristics of acellular dermal matrix (ADM), small intestinal submucosa (SIS) and Bio-Gide scaffolds with acellular vascular matrix (ACVM)-0.25% human-like collagen I (HLC-I) scaffold in tissue engineering blood vessels. The ACVM-0.25% HLC-I scaffold was prepared and compared with ADM, SIS and Bio-Gide scaffolds via hematoxylin and eosin (H&E) staining, Masson staining and scanning electron microscope (SEM) observations. Primary human gingival fibroblasts (HGFs) were cultured and identified. Then, the experiment was established via the seeding of HGFs on different scaffolds for 1, 4 and 7 days. The compatibility of four different scaffolds with HGFs was evaluated by H&E staining, SEM observation and Cell Counting Kit-8 assay. Then, a series of experiments were conducted to evaluate water absorption capacities, mechanical abilities, the ultra-microstructure and the cytotoxicity of the four scaffolds. The ACVM-0.25% HLC-I scaffold was revealed to exhibit the best cell proliferation and good cell architecture. ADM and Bio-Gide scaffolds exhibited good mechanical stability but cell proliferation was reduced when compared with the ACVM-0.25% HLC-I scaffold. In addition, SIS scaffolds exhibited the worst cell proliferation. The ACVM-0.25% HLC-I scaffold exhibited the best water absorption, followed by the SIS and Bio-Gide scaffolds, and then the ADM scaffold. In conclusion, the ACVM-0.25% HLC-I scaffold has good mechanical properties as a tissue engineering scaffold and the present results suggest that it has better biological characterization when compared with other scaffold types.

Identification of novel fibroblast-like cells from stem cells from human exfoliated deciduous teeth.

This study aimed to differentiate and characterize fibroblast-like cells from stem cells from human exfoliated deciduous teeth (SHED). The differentiation of fibroblast-like cells from SHED was carried out by using specific human recombinant connective tissue growth factor (CTGF). To characterize fibroblastic differentiation, the induced cells were subjected to morphological changes, proliferation rate, gene expression analysis using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), flow cytometry, and immunofluorescence staining. The commercial primary human gingival fibroblasts served as positive control in this study. The results from characterization analysis were compared with that of commercial cells to ensure that the cells differentiated from SHED were fibroblast-like cells. The results showed the inductive effect of CTGF for fibroblastic differentiation in SHED. SHED-derived fibroblasts were successfully characterized despite having similar morphological appearance, i.e., (i) significant proliferation rate between fibroblast-like cells and SHED, (ii) high expression of fibroblast-associated markers in qRT-PCR analysis, and (iii) positive staining against collagen type 1, fibroblast-specific protein 1, and human thymic fibroblasts in flow cytometry analysis and immunofluorescence staining. The same expression patterns were found in primary human gingival fibroblasts, respectively. SHED as negative control showed lower expression or no signal, thus confirming the cells differentiated from SHED were fibroblast-like cells. Taken together, the protocol adopted in this study suggests CTGF to be an appropriate inducer in the differentiation of SHED into fibroblast-like cells. The fibroblast-like cells differentiated from SHED could be used in future in vitro and in vivo dental tissue regeneration studies as well as in clinical applications where these cells are needed.

Effect of Sodium Lauryl Sulfate (SLS) on Primary Human Gingival Fibroblasts in an In Vitro Wound Healing Model.

Sodium lauryl sulfate (SLS) is a surfactant used to decrease the surface tension of water. Most commercially available dentifrices contain 0.5-2.0% SLS. This study investigated the potential effect of SLS on oral wound healing using primary human gingival fibroblasts (HGFs). HGFs cells were grown in12-well culture plates in DMEM medium. A 3 mm wound was created on confluent HGFs. The cells were challenged with 0 (the control group), 0.01, 0.02, 0.03, 0.04, or 0.05% SLS-containing media once daily for 2 minutes. The cells were stained on day 0, 2, 4, 6 and 8. The percent of wound fill area was measured. On day 2, 4, 6, and 8, the wound fill of the control group (0% SLS) was 15, 35, 67 and 98%, respectively; at 0.01% SLS, it was 10, 20, 65 and 84%; at 0.02%, it was 7, 10, 15 and 25%; at 0.03% SLS, it was only 5% and 8% on day 2 and 4. Our results showed a dose- and time-dependent inhibition on HGFs wound fill by SLS; however, future in vivo studies are needed to validate if our in vitro findings using SLS-free dentifrices to avoid the potential delay of wound healing.

Enhancing adhesion and alignment of human gingival fibroblasts on dental implants

BackgroundPromoting the directional attachment of gingiva to the dental implant leads to the formation of tight connective tissue which acts as a seal against the penetration of oral bacteria. Such a directional growth is mostly governed by the surface texture. Material and methodsIn this study, three different methods, mechanical structuring, chemical etching and laser treatment, have been explored for their applicability in promoting cellular attachment and alignment of human primary gingival fibroblasts (HGFIBs). ResultsThe effectiveness of mechanical structuring was shown as a simple and a cost-effective method to create patterns to align HGIFIBs. ConclusionCombining mechanical structuring with chemical etching enhanced both cellular attachment and the cellular alignment.

Relevance of Topographic Parameters on the Adhesion and Proliferation of Human Gingival Fibroblasts and Oral Bacterial Strains.

Dental implantology allows replacement of failing teeth providing the patient with a general improvement of health. Unfortunately not all reconstructions succeed, as a consequence of the development of infections of bacterial origin on the implant surface. Surface topography is known to modulate a differential response to bacterial and mammalian cells but topographical measurements are often limited to vertical parameters. In this work we have extended the topographical measurements also to lateral and hybrid parameters of the five most representative implant and prosthetic component surfaces and correlated the results with bacterial and mammalian cell adhesion and proliferation outcomes. Primary human oral gingival fibroblast (gum cells) and the bacterial strains: Streptococcus mutans, Streptococcus sanguinis and Aggregatibacter actinomycetemcomitans, implicated in infectious processes in the oral/implant environment were employed in the presence or absence of human saliva. The results confirm that even though not all the measured surface is available for bacteria to adhere, the overall race for the surface between cells and bacteria is more favourable to the smoother surfaces (nitrided, as machined or lightly acid etched) than to the rougher ones (strong acid etched or sandblasted/acid etched).

Monitoring of Virulence Factors and Metabolic Activity in Aggregatibacter Actinomycetemcomitans Cells Surviving Antimicrobial Photodynamic Therapy via Nano-Chitosan Encapsulated Indocyanine Green

Aggregatibacter actinomycetemcomitans is an etiological agent that is frequently found in both chronic and aggressive periodontitis as well as peri-implantitis. In this study we evaluated the effects of antimicrobial photodynamic therapy (aPDT) as an alternative treatment modality by nano-chitosan encapsulated indocyanine green (CNPs/ICG) as a photosensitizer on the virulence features of cell-surviving aPDT against A. actinomycetemcomitans. Cell cytotoxicity effect of CNPs/ICG was evaluated on primary human gingival fibroblast cells. A. actinomycetemcomitans ATCC 33384 photosensitized with CNPs/ICG were irradiated with diode laser at wavelength of 810 nm for 1 min, following bacterial viability measurements. The biofilm formation ability, metabolic activity and antimicrobial susceptibility profiles were assessed for cell-surviving aPDT. The effect of aPDT on the expression of virulent gene (fieF), encoding Ferrous-iron efflux pump, was evaluated by quantitative real-time PCR. CNPs/ICG-aPDT resulted in the significant reduction of cell viability (91%), the biofilm formation capacity (53%), and metabolic activity (48%) of A. actinomycetemcomitans when compared to the control group (P < 0.05); As well as, fieF gene expression was downregulated (14.8-fold) after the strains were treated with aPDT. The virulence of A. actinomycetemcomitans strain is reduced in cells surviving aPDT with CNPs/ICG, and this may have potential implications of aPDT for the treatment of A. actinomycetemcomitans infections in periodontitis and peri-implantitis in vivo.

In situ photochemical crosslinking of hydrogel membrane for Guided Tissue Regeneration

ObjectivePeriodontitis is an inflammatory disease that destroys the tooth-supporting attachment apparatus. Guided tissue regeneration (GTR) is a technique based on a barrier membrane designed to prevent wound space colonization by gingival cells. This study examined a new formulation composed of two polymers that could be photochemically cross-linked in situ into an interpenetrated polymer network (IPN) forming a hydrogel membrane. MethodsWe synthetized and characterized silanized hydroxypropyl methylcellulose (Si-HPMC) for its cell barrier properties and methacrylated carboxymethyl chitosan (MA-CMCS) for its degradable backbone to use in IPN. Hydrogel membranes were cross-linked using riboflavin photoinitiator and a dentistry visible light lamp. The biomaterial’s physicochemical and mechanical properties were determined. Hydrogel membrane degradation was evaluated in lysozyme. Cytocompatibility was estimated by neutral red uptake. The cell barrier property was studied culturing human primary gingival fibroblasts or human gingival explants on membrane and analyzed with confocal microscopy and histological staining. ResultsThe IPN hydrogel membrane was obtained after 120s of irradiation. The IPN showed a synergistic increase in Young moduli compared with the single networks. The CMCS addition in IPN allows a progressive weight loss compared to each polymer network. Cytocompatibility was confirmed by neutral red assay. Human cell invasion was prevented by hydrogel membranes and histological sections revealed that the biomaterial exhibited a barrier effect in contact with soft gingival tissue. SignificanceWe demonstrated the ability of an innovative polymer formulation to form in situ, using a dentist’s lamp, an IPN hydrogel membrane, which could be an easy-to-use biomaterial for GTR therapy.

Activity of two hyaluronan preparations on primary human oral fibroblasts.

Background and ObjectiveThe potential benefit of using hyaluronan (HA) in reconstructive periodontal surgery is still a matter of debate. The aim of the present study was to evaluate the effects of two HA formulations on human oral fibroblasts involved in soft tissue wound healing/regeneration.Material and MethodsMetabolic, proliferative and migratory abilities of primary human palatal and gingival fibroblasts were examined upon HA treatment. To uncover the mechanisms whereby HA influences cellular behavior, wound healing‐related gene expression and activation of signaling kinases were analyzed by qRT‐PCR and immunoblotting, respectively.ResultsThe investigated HA formulations maintained the viability of oral fibroblasts and increased their proliferative and migratory abilities. They enhanced expression of genes encoding type III collagen and transforming growth factor‐β3, characteristic of scarless wound healing. The HAs upregulated the expression of genes encoding pro‐proliferative, pro‐migratory, and pro‐inflammatory factors, with only a moderate effect on the latter in gingival fibroblasts. In palatal but not gingival fibroblasts, an indirect effect of HA on the expression of matrix metalloproteinases 2 and 3 was detected, potentially exerted through induction of pro‐inflammatory cytokines. Finally, our data pointed on Akt, Erk1/2 and p38 as the signaling molecules whereby the HAs exert their effects on oral fibroblasts.ConclusionBoth investigated HA formulations are biocompatible and enhance the proliferative, migratory and wound healing properties of cell types involved in soft tissue wound healing following regenerative periodontal surgery. Our data further suggest that in gingival tissues, the HAs are not likely to impair the healing process by prolonging inflammation or causing excessive MMP expression at the repair site.

Micro-/nano-topography of selective laser melting titanium enhances adhesion and proliferation and regulates adhesion-related gene expressions of human gingival fibroblasts and human gingival epithelial cells.

BackgroundSelective laser melting (SLM) titanium is an ideal option to manufacture customized implants with suitable surface modification to improve its bioactivity. The peri-implant soft tissues form a protective tissue barrier for the underlying osseointegration. Therefore, original microrough SLM surfaces should be treated for favorable attachment of surrounding soft tissues.Material and methodsIn this study, anodic oxidation (AO) was applied on the microrough SLM titanium substrate to form TiO2 nanotube arrays. After that, calcium phosphate (CaP) nanoparticles were embedded into the nanotubes or the interval of nanotubes by electrochemical deposition (AOC). These two samples were compared to untreated (SLM) samples and accepted mechanically polished (MP) SLM titanium samples. Scanning electron microscopy, energy dispersive spectrometry, X-ray diffraction, surface roughness, and water contact angle measurements were used for surface characterization. The primary human gingival epithelial cells (HGECs) and human gingival fibroblasts (HGFs) were cultured for cell assays to determine adhesion, proliferation, and adhesion-related gene expressions.ResultsFor HGECs, AOC samples showed significantly higher adhesion, proliferation, and adhesion-related gene expressions than AO and SLM samples (P<0.05) and similar exceptional ability in above aspects to MP samples. At the same time, AOC samples showed the highest adhesion, proliferation, and adhesion-related gene expressions for HGFs (P<0.05).ConclusionBy comparison between each sample, we could confirm that both anodic oxidation and CaP nanoparticles had improved bioactivity, and their combined utilization may likely be superior to mechanical polishing, which is most commonly used and widely accepted. Our results indicated that creating appropriate micro-/nano-topographies can be an effective method to affect cell behavior and increase the stability of the peri-implant mucosal barrier on SLM titanium surfaces, which contributes to its application in dental and other biomedical implants.

High glucose-induced oxidative stress impairs proliferation and migration of human gingival fibroblasts.

Delayed gingival wound healing is widely observed in periodontal patients with diabetes. However, the molecular mechanisms of the impaired function of gingival fibroblasts in diabetes remain unclear. The purpose of this study was to investigate changes in the properties of human gingival fibroblasts (HGFs) under high-glucose conditions. Primary HGFs were isolated from healthy gingiva and cultured with 5.5, 25, 50, and 75 mM glucose for 72 h. In vitro wound healing, 5-ethynyl-2′-deoxyuridine (EdU), and water-soluble tetrazolium salt (WST-8) assays were performed to examine cell migration and proliferation. Lactase dehydrogenase (LDH) levels were measured to determine cytotoxicity. The mRNA expression levels of oxidative stress markers were quantified by real-time PCR. Intracellular reactive oxygen species (ROS) were also measured in live cells. The antioxidant N-acetyl-l-cysteine (NAC, 1 mM) was added to evaluate the involvement of ROS in the glucose effect on HGFs. As a result, the in vitro wound healing assay showed that high glucose levels significantly reduced fibroblast migration and proliferation at 6, 12, 24, 36, and 48 h. The numbers of cells positive for EdU staining were decreased, as was cell viability, at 50 and 75 mM glucose. A significant increase in LDH was proportional to the glucose concentration. The mRNA levels of heme oxygenase-1 and superoxide dismutase-1 and ROS levels were significantly increased in HGFs after 72 h of exposure to 50 mM glucose concentration. The addition of NAC diminished the inhibitory effect of high glucose in the in vitro wound healing assay. The results of the present study show that high glucose impairs the proliferation and migration of HGFs. Fibroblast dysfunction may therefore be caused by high glucose-induced oxidative stress and may explain the delayed gingival wound healing in diabetic patients.

Exome Sequencing Discloses Ionizing-radiation-induced DNA Variants in the Genome of Human Gingiva Fibroblasts.

Ionizing radiation can induce genomic lesions such as DNA double-strand breaks whose incomplete or faulty repair can result in mutations, which in turn can influence cellular functions and alter the fate of affected cells and organ systems. Ionizing-radiation-induced sequence alterations/mutations occur in a stochastic manner, which contributes to an increased cancer risk in irradiated individuals. Ionizing radiation exposure, and particularly acute doses at high dose rates (as often observed in radiation accidents), induce alterations in the genome that in part will reflect specific characteristics of the DNA damage response and the repair mechanisms involved. Here, the exome of primary human gingival fibroblasts not exposed or exposed to 0.2, 2, 5, or 10 Gy of x rays was investigated after 16 h of DNA repair for ionizing-radiation-induced mutations. The irradiation effect with varying dose was investigated using three different bioinformatic filters for the analysis of accumulated variants per Mb of genomic DNA and per cytogenetic bands. A highly stringent cutoff of 20-fold coverage was used for all analyses. Comparing exome DNA from irradiated and nonirradiated cells disclosed a characteristic variation of the frequency of ionizing-radiation-induced single-nucleotide variants as well as small insertions and deletions among chromosomes and their subregions. Increases in ionizing-radiation-induced variants with increasing dose were highly significant (p = 2.2 × 10, Kruskal-Wallis test). These results indicate that certain chromosomal regions may be more prone to accumulating particular ionizing-radiation-induced alterations than others, which points to a characteristic metasignature in the irradiated exome.

Curcumin inhibits TGF-β1-induced connective tissue growth factor expression through the interruption of Smad2 signaling in human gingival fibroblasts.

Many fibrotic processes are associated with an increased level of transforming growth factor-β1 (TGF-β1). TGF-β1 can increase synthesis of matrix proteins and enhance secretion of protease inhibitors, resulting in matrix accumulation. Connective tissue growth factor (CTGF) is a downstream profibrotic effector of TGF-β1 and is associated with the fibrosis in several human organs. Curcumin has been applied to reduce matrix accumulation in fibrotic diseases. This study was aimed to evaluate whether curcumin could suppress TGF-β1-induced CTGF expression and its related signaling pathway involving in this inhibitory action in primary human gingival fibroblasts. The differences in CTGF expression among three types of gingival overgrowth and normal gingival tissues were assessed by immunohistochemistry. Gingival fibroblast viability in cultured media with different concentrations of curcumin was studied by MTT assay. The effect of curcumin on TGF-β1-induced CTGF expression in primary human gingival fibroblasts was examined by immunoblotting. Moreover, the proteins involved in TGF-β1 signaling pathways including TGF-β1 receptors and Smad2 were also analyzed by immunoblotting. CTGF was highly expressed in fibroblasts, epithelial cells and some of endothelial cells, smooth muscle cells, and inflammatory cells in phenytoin-induced gingival overgrowth tissues rather than in those of hereditary and inflammatory gingival overgrowth tissues. Moreover, CTGF expression in the epithelial and connective tissue layers was higher in phenytoin-induced gingival overgrowth tissues than in normal gingival tissues. Curcumin was nontoxic and could reduce TGF-β1-induced CTGF expression by attenuating the phosphorylation and nuclear translocation of Smad2. Curcumin can suppress TGF-β1-induced CTGF expression through the interruption of Smad2 signaling.

Biological evaluation of subgingivally placed direct resin composite materials.

Placement of composite resin restorations in deep subgingival cavities can damage surrounding soft tissues. In addition, commonly used resin-based composites (RBCs) might interfere with wound healing and periodontal health. To clarify cellular interactions with RBCs, we used an MTT assay to investigate adhesion of primary human gingival fibroblasts and human osteoblasts (hFOB 1.19) on five RBC materials with and without surface modifications (alumina blasting with 50- or 110-μm Al2O3). In addition, high-performance liquid chromatography (HPLC) was used to determine release of resin monomers from RBCs after 1 h, 1 day, and 7 days. As compared with tissue culture plastics (the control), cellular adhesion was significantly lower (P < 0.001) for human gingival fibroblasts and osteoblasts. Only minor, nonsignificant differences between individual RBCs were identified. HPLC analyses identified the release of three bifunctional methacrylates bisphenol A glycerolate dimethacrylate, triethylene glycol dimethacrylate, and diurethane dimethacrylate from RBCs and showed that monomer release increased between 1 h and 1 day but remained low. The present findings suggest that surface adhesion in the subgingival area is limited for the tested RBCs. Although residual monomer release was low for all tested RBCs, it might be sufficient to adversely affect cell adhesion.

Live cell imaging reveals different modes of cytotoxic action of extracts derived from commonly used luting cements

ObjectiveTo compare cytotoxicity of extracts derived from commonly used luting cements: Hoffmann’s Zinc Phosphate (ZPC), GC Fuji Plus Resin Modified Glass Ionomer (RMGIC) and 3M ESPE RelyX Unicem Resin Cement (RC) on primary human gingival fibroblasts (HGFs). DesignHGFs were exposed to different concentrations of the ZPC, RMGIC and RC extracts. The cytotoxicity was assessed with the PrestoBlue Cell Viability Reagent and viable cells were counted by a haemocytometer using the trypan blue exclusion test. In order to determine the primary mechanism of the cell death induced by extracts from different luting cements, the real-time monitoring of caspase-3/-7 activity and membrane integrity of cells was employed. ResultsThe extracts from the RMGIC and ZPC decreased the metabolic activity and numbers of viable cells. Unexpectedly, the extracts from the RC evoked only small effects on the metabolic activity of HGFs with a decreasing number of viable cells in a dose-and time-dependent manner. The live cell imaging revealed that the apoptosis was the primary mechanism of a cell death induced by the extracts derived from the RMGIC, whereas the extracts from the RC and ZPC induced a cell death through a necrotic and caspase-independent pathway. ConclusionsThe apoptosis was the primary mechanism of the cell death induced by the extracts derived from the RMGIC, whereas the extracts from the RC and ZPC induced a cell death via a necrotic pathway. We suggest that metabolic assays commonly used to assess the cytotoxicity of luting cements should be validated by alternative methods.

Biological effects of Er:YAG laser irradiation on the proliferation of primary human gingival fibroblasts.

We investigated the biological effects of Er:YAG laser (2940-nm; DELight, HOYA ConBio, Fremont, California) irradiation at fluences of 3.6, 4.2, 4.9, 6.3, 8.1 or 9.7 J cm-2 at 20 or 30 Hz for 20 or 30 seconds on primary human gingival fibroblasts (HGFs). Irradiation at 6.3 J cm-2 promoted maximal cell proliferation, determined by WST-8 assay and crystal violet staining, but was accompanied by lactate dehydrogenase release, on day 3 post-irradiation. Elevation of ATP level, Ki67 staining, and cyclin-A2 mRNA expression confirmed that Er:YAG affected the cell cycle and increased the number of proliferating cells. Transmission electron microscopy showed alterations of mitochondria and ribosomal endoplasmic reticulum (ER) at 3 hours post-irradiation at 6.3 J cm-2 , and the changes subsided after 24 hours, suggesting transient cellular injury. Microarray analysis revealed up-regulation of 21 genes involved in heat-related biological responses and ER-associated degradation. The mRNA expression of heat shock protein 70 family was increased, as validated by Real-time PCR. Surface temperature measurement confirmed that 6.3 J cm-2 generated heat (40.9°C post-irradiation). Treatment with 40°C-warmed medium increased proliferation. Laser-induced proliferation was suppressed by inhibition of thermosensory transient receptor potential channels. Thus, despite causing transient cellular damage, Er:YAG laser irradiation at 6.3 J cm-2 strongly potentiated HGF proliferation via photo-thermal stress, suggesting potential wound-healing benefit.

Graphene oxide improves the biocompatibility of collagen membranes in an in vitro model of human primary gingival fibroblasts

Commercial collagen membranes are used in oral surgical procedures as scaffolds for bone deposition in guided bone regeneration. Here, we have enriched them with graphene oxide (GO) via a simple non-covalent functionalization, exploiting the capacity of oxygenated carbon functional moieties of GO to interact through hydrogen bonding with collagen. In the present paper, the GO-coated membranes have been characterized in terms of stability, nano-roughness, biocompatibility and induction of inflammatory response in human primary gingival fibroblast cells. The obtained coated membranes are demonstrated not to leak GO in the bulk solution, and to change some features of the membrane, such as stiffness and adhesion between the membrane and the atomic force microscopy (AFM) tip. Moreover, the presence of GO increases the roughness and the total surface exposed to the cells, as demonstrated by AFM analyses. The obtained material is biocompatible, and does not induce inflammation in the tested cells.

Nanoscale silver depositions inhibit microbial colonization and improve biocompatibility of titanium abutments

Although titanium dental implants are biocompatible, exhibit excellent corrosion resistance and high mechanical resistance, the material fails in providing resistance to infection because it exhibits poor antimicrobial activity. To address these issues, we deposited silver onto titanium abutments (Grade 5 titanium discs) using direct current (DC) sputtering and assessed the antimicrobial activity and biocompatibility of the modified implant material. Atomic absorption spectrometry and X-ray photoelectron spectroscopy were employed to investigate the concentration and elemental composition of the deposited silver. As expected, silver deposited using DC plasma was uniform and good control over the deposition could be achieved by varying the sputtering time. Moderate biocompatible responses (up to 69% viability) were observed in primary human gingival fibroblast cells incubated in the presence of Ti sputtered with Ag for 5min. Silver deposited titanium (Ti-Ag) showed excellent antibacterial effects on Pseudomonas aeruginosa and Streptococcus mutans at a very low concentration (Ag content 1.2 and 2.1μg/mm2). However, higher concentration of silver (6μg/mm2) was required to achieve a reduction in cell viability of Staphylococcus aureus and Candida albicans. The silver sputtered Ti abutments could maintain a long-term antibacterial activity as evidenced by the release of silver up to 22days in simulated body fluid. Our study illustrates that silver deposited titanium is indeed a promising candidate for soft tissue integration on dental abutments and prevents initial microbial adhesion.