Migration Of Human Gingival Fibroblasts Research Articles

Enhanced functionality and migration of human gingival fibroblasts on vacuum ultraviolet light-treated titanium: An implication for mitigating cellular stress to improve peri-implant cellular reaction.

The maintenance of peri-implant health relies significantly on the integrity of the peri-implant seal, particularly vulnerable at the interface between implant abutment and soft tissue. Early healing stages around implants involve cellular exposure to oxidative stress. This study aimed to investigate whether vacuum ultraviolet (VUV)-treated titanium augments the growth and functionality of human gingival fibroblasts while mitigating cellular stress. Machined titanium plates underwent treatment with 172 nm VUV light for one minute, with untreated plates as controls. Human gingival fibroblasts were cultured on treated and untreated plates, and their behavior, growth, and functionality were assessed. Functionally impaired fibroblasts, treated with hydrogen peroxide, were also cultured on these titanium plates, and plate-to-plate transmigration ability was evaluated. Fibroblasts on VUV-treated titanium exhibited a 50% reduction in intracellular reactive oxygen species production compared to controls. Additionally, glutathione, an antioxidant, remained undepleted in cells on VUV-treated titanium. Furthermore, the expression levels of inflammatory cytokines IL-1β and IL-8 decreased by 40-60% on VUV-treated titanium. Consequently, fibroblast attachment and proliferation doubled on VUV-treated titanium compared to those in the controls, leading to enhanced cell retention. Plate-to-plate transmigration assays demonstrated that fibroblasts migrated twice as far on VUV-treated surfaces compared to those in the controls. In particular, the transmigration ability, impaired in functionally impaired fibroblasts on the controls, was preserved on VUV-treated titanium. VUV-treated titanium promotes the growth, function, and migration of human gingival fibroblasts by reducing cellular stress and enhancing antioxidative capacity. Notably, the transmigration ability significantly improved on VUV-treated titanium.

Novel approach to soft tissue regeneration: in vitro study of compound hyaluronic acid and horizontal platelet-rich fibrin combination.

This study aims to develop a compound biomaterial to achieve effective soft tissue regeneration. Compound hyaluronic acid (CHA) and liquid horizontal-platelet-rich fibrin (H-PRF) were mixed at a ratio of 1:1 to form a CHA-PRF gel. Human gingival fibroblasts (HGFs) were used in this study. The effect of CHA, H-PRF, and the CHA-PRF gel on cell viability was evaluated by CCK-8 assays. Then, the effect of CHA, H-PRF, and the CHA-PRF gel on collagen formation and deposition was evaluated by qRT‒PCR and immunofluorescence analysis. Finally, qRT‒PCR, immunofluorescence analysis, Transwell assays, and scratch wound-healing assays were performed to determine how CHA, H-PRF, and the CHA-PRF gel affect the migration of HGFs. The combination of CHA and H-PRF shortened the coagulation time of liquid H-PRF. Compared to the pure CHA and H-PRF group, the CHA-PRF group exhibited the highest cell proliferation at all time points, as shown by the CCK-8 assay. Col1a and FAK were expressed at the highest levels in the CHA-PRF group, as shown by qRT‒PCR. CHA and PRF could stimulate collagen formation and HGF migration, as observed by fluorescence microscopy analysis of COL1 and F-actin and Transwell and scratch healing assays. The CHA-PRF group exhibited greater potential to promote soft tissue regeneration by inducing cell proliferation, collagen synthesis, and migration in HGFs than the pure CHA or H-PRF group. CHA-PRF can serve as a great candidate for use alone or in combination with autografts in periodontal or peri-implant soft tissue regeneration.

Comparative effect of anthocyanin on proliferation and migration of human gingival fibroblasts in the absence or presence of nicotine.

Oral fibroblast malfunction can result in periodontal diseases. Nicotine can prolong the healing process as an irritant of oral tissues. Anthocyanins have been demonstrated to have potential benefits in preventing or treating smoking-related periodontal diseases. Cyanidin chloride's (CC's) potential in oral wound healing and the viability, proliferation, and migration of human gingival fibroblasts (HGFs) were examined in the presence and absence of nicotine by an in vitro study. The effects of different nicotine concentrations (1, 2, 3, 4, and 5 mM) on the viability and proliferation of HGF cells were evaluated in the presence and absence of different CC concentrations (5, 10, 25, and 50 μM) using the quantitative MTT assay. The scratch test was performed to evaluate the migration of CC-treated cells in the presence of 2.5-mM nicotine. No cytotoxicity was observed at 1‒100 μM CC concentrations after 24, 48, and 72 hours of exposure to HGF cells. However, a concentration of 200 μM significantly reduced cell viability by about 20% at all the three-time intervals (P<0.05). Also, 3‒5-mM concentrations of nicotine significantly reduced cell viability in a dose- and time-dependent manner. Moreover, the understudied CC concentrations decreased nicotine's adverse effects on cell migration to some extent. Although the understudied CC concentrations could not significantly reduce the adverse effects of understudied nicotine concentrations on the viability and proliferation of HGF cells, they were able to reduce the detrimental effects of nicotine on cell migration significantly.

Photobiomodulation effects of pulsed and continuous wave near-infrared laser on the proliferation and migration of human gingival fibroblasts: An in vitro study.

There are limited data on comparison of pulsed and continuous wave in photobiomodulation therapy (PBM). This study aimed to investigate the effect of PBM with 980 nm laser in pulsed and continuous wave on the proliferation and migration of human gingival fibroblasts (HGF) cells. Cultured HGF were divided into three main groups: (1) irradiated in pulsed mode (frequencies of 50 and 25 KHz; energy densities of 3 and 5 J/cm2 ), (2) irradiated in continuous mode (energy densities of 3.2 and 5.2 J/cm2 ), and (3) no irradiation as control group. HGF proliferation rate was measured by MTT assay at 24, 48, and 72 h post irradiation. In addition, HGF migration rate was measured by scratch test at 24 h post PBM. At 24 h, the group received continuous irradiation at 5.2 J/cm2 showed significantly higher proliferation compared with the control group (p = 0.012). At 48 and 72 h, the groups received continuous, and 50 Hz pulsed irradiation at energy densities of 5.2 and 5 J/cm2 respectively, had significantly higher HGF proliferation rates compared to the control (p < 0.05). Only the continuous irradiations were effective in significant increase of the cell migration. In conclusion, continuous PBM at energy density of 5.2 J/cm2 showed promising effect on HGF proliferation and migration.

Multi-functional nanogel with cascade catalytic performance for treatment of diabetic oral mucosa ulcer.

Introduction: Diabetic oral mucosa ulcers face challenges of hypoxia, hyperglycemia and high oxidative stress, which result in delayed healing process. Oxygen is regarded as an important substance in cell proliferation, differentiation and migration, which is beneficial to ulcer recovery. Methods: This study developed a multi-functional GOx-CAT nanogel (GCN) system for the treatment of diabetic oral mucosa ulcers. The catalytic activity, ROS scavenge and oxygen supply ability of GCN was validated. The therapeutic effect of GCN was verified in the diabetic gingival ulcer model. Results: The results showed that the nanoscale GCN was capable of significantly eliminating intracellular ROS, increasing intracellular oxygen concentration and accelerating cell migration of human gingival fibroblasts, which could promote diabetic oral gingival ulcer healing in vivo by alleviating inflammation and promoting angiogenesis. Discussion: This multifunctional GCN with ROS depletion, continuous oxygen supply and good biocompatibility, which might provide a novel therapeutic strategy for effective treatment of diabetic oral mucosa ulcers.

Nano zinc oxide-functionalized nanofibrous microspheres: A bioactive hybrid platform with antimicrobial, regenerative and hemostatic activities

Bioactive hybrid constructs are at the cutting edge of innovative biomaterials. PLA nanofibrous microspheres (NF-MS) were functionalized with zinc oxide nanoparticles (nZnO) and DDAB-modified nZnO (D-nZnO) for developing inorganic/nano-microparticulate hybrid constructs (nZnO@NF-MS and D-nZnO@NF-MS) merging antibacterial, regenerative, and haemostatic functionalities. The hybrids appeared as three-dimensional NF-MS frameworks made-up entirely of interconnecting nanofibers embedding nZnO or D-nZnO. Both systems achieved faster release of Zn2+ than their respective nanoparticles and D-nZnO@NF-MS exhibited significantly greater surface wettability than nZnO@NF-MS. Regarding bioactivity, D-nZnO@NF-MS displayed a significantly greater and fast-killing effect against Staphylococcus aureus. Both nZnO@NF-MS and D-nZnO@NF-MS showed controllable concentration-dependent cytotoxicity to human gingival fibroblasts (HGF) compared with pristine NF-MS. They were also more effective than pristine NF-MS in promoting migration of human gingival fibroblasts (HGF) in the in vitro wound healing assay. Although D-nZnO@NF-MS showed greater in vitro hemostatic activity than nZnO@NF-MS (blood-clotting index 22.82 ± 0.65% vs.54.67 ± 2.32%), both structures exhibited instant hemostasis (0 s) with no blood loss (0 mg) in the rat-tail cutting technique. By merging the multiple therapeutic bioactivities of D-nZnO and the 3D-structural properties of NF-MS, the innovative D-nZnO@NF-MS hybrid construct provides a versatile bioactive material platform for different biomedical applications.

Evaluation of the wound-healing potential of the kiwifruit extract by assessing its effects on human gingival fibroblasts and angiogenesis.

Oral surgery and surgical interventions in the field of periodontology require the uneventful wound healing of soft tissues to be successful. However, since the oral cavity is exposed to saliva and is a niche for oral microorganisms, wound healing can be delayed, causing pain and discomfort to the patient. Hence, a great deal of interest has shifted to the study of agents that can enhance wound-healing processes. The aim of the present study was to evaluate the oral wound-healing potential of the Actinidia deliciosa (kiwifruit) extract by assessing its effects on the viability, proliferation and migration of human gingival fibroblasts, and its ability to enhance angiogenesis in the chicken chorioallantoic membrane (CAM). The collection of the A. deliciosa extract and experimentation were carried out in the Maratha Mandal Dental College, Belgavi, India. Human gingival fibroblasts were cultured in a suitable medium and treated with 6.25 μg, 12.5 μg or 25 μg of the ethanolic kiwifruit extract. The MTT cell viability and scratch assays were then carried out in vitro. To determine the angiogenic potential of the extract, solutions of concentrations of 10 μg/mL, 50 μg/mL and 100 μg/mL were transferred to sterile Whatman™ filter paper disks, which were placed on the CAM of 5-day-old Leghorn chicken eggs. The ethanolic extract of A. deliciosa was not cytotoxic to gingival fibroblasts at a dosage of 6.25 μg/mL. In addition, the scratch assay demonstrated the migration of the fibroblasts and the closure of an artificially created wound within 24 h, which is similar to in vivo wound closure. The CAM assay showed that the kiwifruit extract concentrations of 10 μg/mL and 50 μg/mL showed a moderate proangiogenic effect, and a concentration of 100 μg/mL exerted a strong pro-angiogenic effect. The results demonstrate that the kiwifruit extract is safe to use, can enhance the proliferation and migration of human gingival fibroblasts, and promotes angiogenesis, making it a suitable oral wound-healing agent.

Comparison of Pulsed and Continuous Wave Diode Laser at 940 nm on the Viability and Migration of Gingival Fibroblasts.

Gingival fibroblasts have critical roles in oral wound healing. Photobiomodulation (PBM) has been shown to promote mucosal healing and is now recommended for managing oncotherapy-associated oral mucositis. This study examined the effects of the emission mode of a 940 nm diode laser on the viability and migration of human gingival fibroblasts. Cells were cultured in a routine growth media and treated with PBM (average power 0.1 W cm-2 , average fluence 3 J cm-2 , every 12 h for six sessions) in one continuous wave and two pulsing settings with 20% and 50% duty cycles. Cell viability was assessed using MTT, and digital imaging quantified cell migration. After 48 and 72 h, all treatment groups had significantly higher viability (n = 6, P < 0.05) compared with the control. The highest viability was seen in the pulsed (20% duty cycle) group at the 72-h time point. PBM improved fibroblast migration in all PBM-treated groups, but differences were not statistically significant (n = 2, P > 0.05). PBM treatments can promote cell viability in both continuous and pulsed modes. Further studies are needed to elucidate the optimal setting for PBM-evoked responses for its rationalized use in promoting specific phases of oral wound healing.

Novel Mg-Ca-La alloys for guided bone regeneration: Mechanical performance, stress corrosion behavior and biocompatibility

Guided bone regeneration (GBR) is a therapeutic method that is used to augment deficient ridges before dental implantation. The non-absorbable membranes need secondary removal surgery, while absorbable collagen membranes fail while being applied to large bone defects because of their relatively low stiffness. The “ideal” membrane for use in GBR has yet to be developed. This study aims to develop and explore the feasibility of Mg-Ca-La alloys for application in GBR. Mg-0.2Ca-0.2La alloy exhibits fine microstructure and a good combination of tensile strength and ductility. Mg-0.2Ca-0.2La alloy is also capable of supporting the adhesion, proliferation and migration of human gingival fibroblasts, and shows good cytocompatibility on MC3T3-E1 cells. The GBR membranes usually need intraoperative bending or shaping to adapt the alveolar ridge. The additional effect of bending stress on magnesium-based materials must be considered due to a well-known issue, namely stress-corrosion cracking (SCC). The SCC results reveal that the bended Mg-0.2Ca-0.2La presents a uniform corrosion morphology with a corrosion rate of 0.09 mm/yr, which is 61% less than that of unstressed ones. Mg-0.2Ca-0.2La alloy possesses adequate strength and ductility, high biocompatibility and slow degradation under bending stress, contributing to adequate malleability and sustained spatial stability for membrane use.

Effects of titanium dioxide microparticles and nanoparticles on cytoskeletal organization, cell adhesion, migration, and proliferation in human gingival fibroblasts in the presence of lipopolysaccharide.

Titanium wear particles may participate in the etiology of peri-implantitis. However, the influence of titanium wear particles on biological behavior of human gingival fibroblasts (HGFs) in the presence of LPS is still not clear. The present study demonstrated the effects of titanium dioxide micro- and nanoparticles (TiO2 MPs and NPs) on HGF cell viability, cytoskeletal organization, adhesion, migration, and proliferation in vitro, and LPS was used to mimic the in vivo condition. Primary HGFs were treated with TiO2 MPs (primaryparticle size <5μm, 0.1mg/ml) and NPs (primary particle size <100nm, 0.1mg/ml) with or without 1μg/ml LPS. The effects of TiO2 MPs and NPs on HGFs cell viability was measured by CCK-8 assay. The proliferation of HGF was detected by Ki67 nuclear staining. The confocal laser scanning microscope (CLSM) was used to detect the internalization of TiO2 MPs and NPs in HGFs as well as the arrangement of F-actin, vinculin, and vimentin organization. Wound healing assay and transwell assay were performed to measure the migration of HGFs induced by TiO2 MPs and NPs. Cell adhesion was measured using fibronectin-coated plates. The relative mRNA and protein expression of adhesion relative protein such as focal adhesion kinase (FAK), fibronectin (FN), and type I collagen (COL1) were measured using quantitative RT-PCR and western blot analysis. One-way analysis of variance (ANOVA) and Student's t-test were used to analyze the statistical significance, and p<.05 was considered statistically significant. TiO2 NPs significantly inhibited HGF cell viability, proliferation, and migration compared with TiO2 MPs group and control group. Compared with control group (2.64±0.09), the mean absorbance of the cells in 1mg/ml TiO2 MPs group and 0.25mg/ml TiO2 NPs group were significantly decreased to 1.93±0.33 (p<.05) and 2.22±0.18 (p<.01), respectively. The cytoskeleton disruption was found in TiO2 NPs group. The mRNA and protein expression were significantly downregulated by TiO2 NPs. Furthermore, both TiO2 NPs and MPs induced more adverse effects on HGFs in the presence of LPS. Our results indicate that TiO2 NPs but not TiO2 MPs significantly disrupt the cytoskeletal organization and inhibited cell adhesion, migration, and proliferation of HGFs. However, in the presence of LPS, TiO2 MPs, and TiO2 NPs enhance these negative effects in HGFs. Titanium wear particles are probably involved in the initiation and progression of peri-implant diseases.

The efficiency of natural wound healing and bacterial biofilm inhibition of Aloe vera and Sodium Chloride toothpaste preparation

BackgroundPrevention is a preliminary focus of periodontitis treatment. Rather than giving complicated treatment to a periodontitis patient, a variety of toothpastes have been suggested to prevent periodontal disease progression. Herbal toothpastes containing natural plant components for maintaining or increasing healing might be a treatment modality for improving oral hygiene. Aloe vera is a medicinal plant with active ingredients that have antioxidant and anti-inflammatory effects. Additionally, increased sodium in the environment inhibits microorganism growth. A toothpaste containing salt and aloe vera may be an option to provide good oral hygiene.AimTo assess the in vitro cell migration of human gingival fibroblasts and antimicrobial effects of an herbal toothpaste containing A. vera and Sodium chloride.MethodsThe cytotoxicity of 0.02% or 0.2% toothpaste solution on human gingival fibroblast cell line was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The cell migration after treatment with 0.2% (v/v) toothpaste was determined using a Boyden Chamber assay. The effect of the toothpaste on inhibiting Porphylomonas gingivalis planktonic and biofilm growth was compared with Chlohexidine (CHX) using a Disk Diffusion and Biofilm susceptibility test, respectively. The results of the cytotoxicity assay, inhibition zone and percentage of live cells in the biofilm were statistically analyzed with One-way analysis of variance. Cell migration and biofilm inhibition were evaluated using the independent sample t-test and multiple t-test, respectively (p = 0.05).ResultsNeither test concentration of the toothpaste solution was toxic to the target cells. The 0.2% concentration was selected for the cell migration experiment. The herbal toothpaste formulation significantly increased cell migration compared with the control group (culture medium) (p = .02) The antimicrobial effect of this formulation on the P. gingivalis planktonic form was lower compared with 0.12% CHX (positive control group), however, it demonstrated greater P. gingivalis biofilm formation inhibition compared with the 0.12% CHX group.ConclusionsThe alternative use of an herbal toothpaste instead of a non-herbal toothpaste formulation should be considered for promoting oral health care. However, further clinical studies are necessary before it can be considered for patient use.

Metformin accelerates wound healing by Akt phosphorylation of gingival fibroblasts in insulin-resistant prediabetes mice.

This study aimed to investigate the effects of metformin on gingival wound healing in insulin-resistant prediabetes. C57BL/6J mice were fed normal diet (ND) or high-fat diet (HFD) for 10weeks; half of the HFD mice were treated with metformin (HFD+ Met) for the last 2weeks. Insulin and glucose tolerance tests were performed. The palatal gingiva (2.0×0.5mm) was surgically removed adjacent to the maxillary molars. Post-surgical wound closure was histomorphometrically evaluated for 1week. The mRNA expression of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in the tissue were quantified by real-time polymerase chain reaction. In vitro, the proliferation and migration of human gingival fibroblasts (HGFs) cultured under high-glucose or control conditions with/without metformin were analyzed. Akt phosphorylation and VEGF expression following the insulin stimulation were evaluated with/without metformin in high-glucose or control media. HFD mice showed significantly higher plasma glucose levels and insulin resistance than ND mice. Gingival wound healing was delayed in HFD group compared with ND group but significantly improved in HFD + MET group. The decreased expression of VEGF and eNOS in HFD group was significantly elevated in the HFD + MET group. The proliferation and migration of HGFs were significantly impaired in high-glucose conditions compared with control; metformin treatment partially attenuated these effects. Metformin treatment significantly recovered the downregulated Akt phosphorylation and VEGF expression in high-glucose conditions. Metformin improved delayed gingival wound healing in insulin-resistant prediabetes by accelerating HGFs proliferation and migration via Akt phosphorylation in insulin signaling pathway.

Improved response of human gingival fibroblasts to titanium coated with micro-/nano-structured tantalum

ObjectivesThis study aims to evaluate the ability of tantalum-coated titanium to improve human gingival fibroblasts’ adhesion, viability, proliferation, migration performance, and the potential molecular mechanisms.Materials and methodsTitanium plates were divided into two groups: (1) no coating (Ti, control), (2) Tantalum-coated titanium (Ta-coated Ti). All samples were characterized by scanning electronic microscopy, surface roughness, and hydrophilicity. Fibroblasts’ performance were analyzed by attached cell number at 1 h, 4 h, and 24 h, morphology at 1 h and 4 h, viability at 1 day, 3 days, 5 days, and 7 days, recovery after wounding at 6 h, 12 h, and 24 h. RT-PCR, western blot were applied to detect attachment-related genes’ expression and protein synthesis at 4 h and 24 h. Student’s t test was used for statistical analysis.ResultsTantalum-coated titanium demonstrates a layer of homogeneously distributed nano-grains with mean diameter of 25.98 (± 14.75) nm. It was found that after tantalum deposition, human gingival fibroblasts (HGFs) adhesion, viability, proliferation, and migration were promoted in comparison to the control group. An upregulated level of Integrin β1 and FAK signaling was also detected, which might be the underlying mechanism.ConclusionIn the present study, adhesion, viability, proliferation, migration of human gingival fibroblasts are promoted on tantalum-coated titanium, upregulated integrin β1 and FAK might contribute to its superior performance, indicating tantalum coating can be applied in transmucosal part of dental implant.Clinical significanceTantalum deposition on titanium surfaces can promote human gingival fibroblast adhesion, accordingly forming a well-organized soft tissue sealing and may contribute to a successful osseointegration.

Assessment of the Photobiomodulation Effect of a Blue Diode Laser on the Proliferation and Migration of Cultured Human Gingival Fibroblast Cells: A Preliminary In Vitro Study

Introduction: Photobiomodulation therapy (PBM) is emerging as an effective strategy for the management of wound healing. The application of red and near infra-red light sources in laser therapy has been the subject of most researches in recent literature. Considering the lack of sufficient evidence in assessing the blue light in PBM, we aimed to investigate the photobiomodulation effect of a blue diode laser on the proliferation and migration of cultured human gingival fibroblast cells as a preliminary in vitro study. Methods: Human gingival fibroblast cells were irradiated with a blue diode laser at a 445 nm wavelength. Irradiation was done using three different powers of 200 mW (irradiation times of 5, 10,15, and 20 seconds); 300 mW (irradiation times of 5, 10, and 15 seconds); and 400 mW (irradiation times of 5 and 10 seconds). The fibroblast cells without laser exposure were considered as control. After 24 hours of incubation, the MTT assay and the wound scratch test were performed on the cells to investigate the biomodulation effect of the blue laser on the proliferation and migration of the cells respectively. The results were analyzed by one-way ANOVA and a post-hoc Tukey test with a P value <0.05 as a statistical significance level. Results: PBM with blue diode laser at power densities of 400 mW/cm2 with irradiation times of 10 and 15 seconds corresponding to energy densities of 4 and 6 J/cm2 exerted the statistically significant positive effect on both proliferation and migration of gingival fibroblast cells. Conclusion: Considering the encouraging findings of this study, PBM with blue diode laser can promote proliferation and migration of human gingival fibroblasts, the key cells involved in the process of oral wound healing.

Interface effects on regulating the behaviors of human gingival fibroblasts on titanium by Zn-PIII

To understand the interface effects between human gingival fibroblasts (HGFs) and the titanium substrate for soft tissue sealing, zinc (Zn) was incorporated into titanium by plasma immersion ion implantation (PIII) in the work, and the durations of 30, 60 and 90 min were adopted for serials of Zn content on titanium to identify the possible dose-effect relationship. Results showed that the implanted Zn formed metallic Zn and zinc oxide on the surface, and the Zn content increased with the extension of implantation time. The release of Zn ions occurred in the saline solution, but the amounts of released ions were inconsistent with the Zn content of implanted samples and their corrosion behaviors, indicating the different reaction activities among the sample surfaces. Moreover, in vitro studies disclosed that the incorporated samples were beneficial to the adhesion, proliferation, and migration of HGFs with appropriate dose of released Zn ions as well as the surface activities, as the fact it was not a directly proportional dose-effect relationship between released Zn ions and the behaviors of cells. It was suggested that Zn-PIII provided an approach to improve the soft tissue sealing of titanium through interfacial activities of implanted samples to regulate the behaviors of HGFs and the usage of Zn ions followed a synergistic manner.

Enhanced wound healing and osteogenic potential of photodynamic therapy on human gingival fibroblasts.

Photodynamic therapy (PDT) has shown ideal antibacterial effects in clinical treatment of periodontal diseases. However, little is known about the specific potential of PDT on human gingival fibroblasts (HGFs) especially cells in the inflamed state, which may contribute to the repairi of periodontal tissue. The effect of PDT with different concentrations of methylene blue (5 μM, 10 μM, 20 μM) on cell vitality of healthy and inflamed human gingival fibroblasts was evaluated by CCK-8, and cell migration was assessed by cell scratching assay. The gene expression of interleukin-6 (IL-6), interleukin-8 (IL-8), type I collagen (Col I), fibronectin (FN) and basic fibroblast growth factor (bFGF) were measured with real-time fluorescent quantitative polymerase chain reaction. The alkaline phosphatase (ALP) production and alizarin red staining of mineralized nodules in healthy and inflamed human gingival fibroblasts was evaluated to explore the effect on osteogenic differentiation. PDT with relatively low concentration of methylene blue (5 μM) inhibited the cell vitality of inflamed human gingival fibroblasts (I-HGFs) slightly (P < 0.05), but had no adverse effect on healthy human gingival fibroblasts (H-HGFs) (P > 0.05). As the concentration increased, PDT with 20 μM methylene blue had significantly negative effect on both healthy and inflamed cells. Further, PDT with 5 μM methylene blue was observed to be able to promote the migration of HGFs especially the healthy state, and increases the expression of wound healing related genes including IL-6, COL1, FN, bFGF in healthy and inflamed HGFs (P < 0.05). PDT with 5 μM methylene blue was also capable of increasing the production of ALP and mineralized nodules (P < 0.05), although the better effect was observed in the laser treatment group. The relatively low concentration of methylene blue mediated PDT is conducive to the growth of H-HGFs while inhibiting the I-HGFs, and it also has the potential to promote the wound healing and osteogenic related functions of both healthy and inflamed HGFs.

Effects of leukocyte-platelet-rich fibrin and advanced platelet-rich fibrin on the viability and migration of human gingival fibroblasts.

Background:Platelet products play a fundamental role in the process of healing. The new generation of platelet-rich fibrin (PRF), namely advanced PRF (A-PRF), has different biological and mechanical properties compared to those of leukocyte-PRF (L-PRF). This study aimed to compare the effects of L-PRF and A-PRF on the viability and migration of human gingival fibroblasts (HGFs).Materials and Methods:In this in vitro study, the effects of A-PRF and L-PRF on the viability and migration of HGFs after 24 and 48 h were evaluated using the methyl thiazolyl tetrazolium assay. The viability of the negative control culture medium was considered to be 100%. The mean optical density of the test groups was divided by that of the negative control group and reported as percentage. One-way ANOVA was applied to assess the effects of time and type of PRF on the viability and migration of HGFs. Pairwise comparisons were made using the Tukey's test.Results:At 24 h, cell viability in the L-PRF group was significantly higher than that in the A-PRF group (P < 0.05). However, no significant difference was noted between the two groups at 48 h. At 24 h, L-PRF caused significantly higher cell migration compared to the negative control group, whereas at 48 h, both A-PRF and L-PRF significantly increased cell migration compared to the control group.Conclusion:Within the limitations of this study, L-PRF and A-PRF had significant effects on the viability and migration of HGFs. Further studies on these platelet concentrates are warranted.

The Effect of Platelet-Rich Fibrin (PRF), Plasma Rich in Growth Factors (PRGF), and Enamel Matrix Proteins (Emdogain) on Migration of Human Gingival Fibroblasts.

Statement of the Problem: Platelet preparations such as plasma rich in growth factors (PRGF), platelet-rich fibrin (PRF), or enamel matrix proteins (Emdogain) are commonly used for soft and hard tissue regeneration. However, their comparative effectiveness has not been extensively studied, and a consensus has yet to be reached on their efficacy. Purpose:The aim of this in vitro study was to compare the effect of PRF, PRGF, and Emdogain on the proliferation of human gingival fibroblasts (HGF). Materials and Method: Artificial wounds were made in HGF cell culture after the fibroblasts reached nearly 100% of confluence. The study groups included leukocyte and platelet-rich fibrin (LPRF), pure platelet-rich fibrin (PPRF), PRGF and Emdogain. Stained cells were photographed at 48 h and one week and the percent of wound filling was measured. Statistical analysis was performed by one-way ANOVA and p values < 0.05 were considered statistically significant. Results:The highest wound filling percentages at both time intervals were observed in the PPRF group, followed by the PRGF. The lowest percentage of wound healing among test groups was observed in the LPRF while Emdogain yielded modest results. However, statistical analysis showed similar wound healing values in PRGF, PPRF, and Emdogain groups. Conclusion: Within the limitations of this study, PRGF, PPRF, and Emdogain were similarly effective in enhancing the fibroblast proliferation and artificial wound closure.

Effect of physical cues of altered extract media from biodegradable magnesium implants on human gingival fibroblasts

Volume stable barrier membranes made of magnesium are very promising in Guided Bone Regeneration (GBR) to treat periodontal bone defects in dentistry due to their excellent biocompatibility and biodegradability. During the degradation process the cells are exposed to the alteration of various parameters, so called physical cues, involving surface alterations due to the formed corrosion layer and medium alterations arising from the dissolved corrosion products. Cell migration of human gingival fibroblasts (HGF), as a crucial parameter for optimal healing process in GBR, has been investigated on magnesium membranes and revealed that medium alterations by dissolved corrosion products have a higher impact on cell migration than surface alterations. However, the effect of each altered medium parameter on cell migration has not been adequately studied, but their roles are crucial to explain the slower migration rate on magnesium surfaces compared to titanium and tissue culture plastic surfaces. Our study investigates the single effect of Mg2+, Ca2+, H2 and increased osmolality as well as the effect of magnesium extracts, which contain a dynamic mixture of previous parameters on cell migration, proliferation and viability of HGF. We showed that at 75 mM Mg2+ concentration and at 0 mM Ca2+, respectively, the cell migration rate is greatly reduced. In complex magnesium extract media, we found that a temporarily increased ratio of Mg2+ to Ca2+ conditioned a slow HGF migration rate. Based on these findings and the characterization of supernatants from HGF migration assays on Mg membranes, we propose, that the slower migration rate of HGF can be explained by the altered ratio of Mg2+ to Ca2+, caused by increasing concentrations of Mg2+ and decreasing concentrations of Ca2+ in the vicinity of the corroding Mg implant, combined with a constantly increased molecular hydrogen concentration in the supernatant. These results are cell type specific and should be checked carefully, if necessary, for Mg implant performance. Statement of SignificanceThe study is providing a systematic approach to explain the main effects of extract medium parameters (physical cues) such as magnesium or calcium ion concentration, osmolality and dissolved molecular hydrogen and CO2 in cell culture media modified by co-incubating with corroding magnesium implants on the migration rate of human gingival fibroblasts (HGF). This study uncovers for the first time the combinatory effect of slightly increased molecular hydrogen and the change in Mg2+/Ca2+ ratio on HGF cell migration.

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.