Functions Of Human Gingival Fibroblasts Research Articles

Achieving Complete Human Gingival Fibroblast Collagen Coverage on Implant Abutments through Vacuum Ultraviolet (VUV) Photofunctionalization.

The formation of a biological seal between implant abutments and the surrounding soft tissue is a preventive strategy against peri-implantitis. The aim of this study is to test the hypothesis that surfaces of prosthetic implant abutments treated with vacuum ultraviolet (VUV) light enhance the growth and function of human gingival fibroblasts. Implant abutments were treated with 172 nm VUV light for one minute. Untreated abutments were subjected as controls. Their surface properties were characterized using SEM, contact angle measurements, and chemical composition analysis. Human gingival fibroblasts were cultured on both untreated and VUV-treated abutments to evaluate cell attachment, proliferation, distribution, and collagen production. Cell detachment assays were also performed under various mechanical and chemical stimuli. After VUV treatment, implant abutments demonstrated a notable transition from hydrophobic to hydrophilic wettability. Surface element analysis revealed a considerable reduction in surface carbon and increases in oxygen and titanium elements on the VUV-treated surfaces. On day 1 of culture, 3.9 times more fibroblasts attached on VUV-treated abutments than on untreated control abutments. Fibroblastic proliferation increased 1.9-3.1 times on VUV-treated abutments, along with a significant improvement in the distribution of populating cells. Collagen production on VUV-treated abutments increased by 1.5-1.7 times. While untreated abutment surfaces showed voids and limited spread of collagen deposition, dense and full coverage of collagen was observed on VUV-treated abutments, with a great contrast in the challenging axial surface zone. Cell retention against mechanical and chemical detaching stimuli was increased 11.3 and 4.3 times, respectively, by VUV treatment. Treatment of implant abutments with VUV light for one minute resulted in a reduction of surface carbon and a transformation of the surface from hydrophobic to hydrophilic. This led to enhanced attachment, proliferation, and retention of human gingival fibroblasts, along with nearly complete collagen coverage on implant abutments. These in vitro results indicate the promising potential of utilizing VUV photofunctionalized implant abutments to enhance soft tissue reaction and sealing mechanisms.

Human Gingival Fibroblast Growth and Function in Response to Laser-Induced Meso- and Microscale Hybrid Topography on Dental Implant Healing Abutments.

To examine the behavior and function of human gingival fibroblasts growing on healing abutments with or without laser-textured topography. Human primary gingival connective tissue fibroblasts were cultured on healing abutments with machined or laser-textured (Laser-Lok, BioHorizons) surfaces. Cellular and molecular responses were evaluated by a variety of tests, including cell density assay (WST-1), fluorescence microscopy, real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and detachment tests. The machined surface showed monodirectional traces and scratches from milling, whereas the laser-textured surface showed a distinct morphology consisting of monodirectional mesoscale channels (15-μm pitch) and woven oblique microridges formed within the channels. There were no differences in initial fibroblast attachment, subsequent fibroblast proliferation, or collagen production between the machined and laser-textured surfaces. Fibroblasts growing on a laser-textured surface were found to spread in one direction along the mesochannels, while cells growing on machined surfaces tended to spread randomly. Fibroblasts on laser-textured surfaces were 1.8 times more resistant to detachment than those on machined surfaces. An adhesive glycoprotein (fibronectin) and transmembrane adhesion linker gene (integrin β-1) were upregulated on laser-textured surfaces. The increased fibroblast retention, uniform growth, and increased transcription of cell adhesion proteins compellingly explain the enhanced tissue-level response to laser-created and hybrid-textured titanium surfaces. These results provide a cellular and molecular rationale for the tissue reaction to this unique surface; in addition, they support its extended use, from implants and healing abutments to diverse prosthetic components where enhanced soft tissue responses would be desirable.

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.

Clinical and immunological aspects of gingival retraction systems in fixed dental prostheses: A systematic review

Objective To describe the existing knowledge on the efficacy of the different gingival retraction systems (GRSs) in gingival displacement, to know their effects on biological functions of human gingival fibroblasts (HGFs), and on the expression of inflammatory mediators (TNF-α and MCP-1) in gingival crevicular fluid (GCF), and saliva. Methods The protocol used for this systematic review was registered in INPLASY: 202410005. A digital search was performed in the databases PubMed/MEDLINE, Scopus, Science Direct, Web of Science, and Google Scholar of the literature published in the English language in the last 17 years (from December 10th, 2006, to May 15th, 2023), and included retrospective randomized clinical studies, prospective, and in vitro experimental studies. In addition, PRISMA criteria were followed. The methodological validity of the selected articles was assessed using Joanna Briggs Institute (JBI) critical appraisal tool, and the modified Consolidated Standards of Reporting Trials checklist (CONSORT). Results 27 articles published between 2006 and 2023 were evaluated. Six hundred 32 subjects, aged between 18 and 65, participated in the clinical studies. 93.7% of the studies assessed periodontally healthy patients, and only 6.3% evaluated patients with mild gingivitis. Also, 882 teeth were samples, of which the majority were posterior teeth (54%). The most commonly used GRSs was aluminum chloride gingival retraction paste (74%). The GCF samples were taken in 67% of the studies, and ELISA was used in all studies (100%) to determine inflammatory mediators. The most frequently analyzed marker was TNF-α (67%). Conclusion The system Merocel Strips (Mystic, conn, USA) achieved the highest level of gingival displacement (1.66 ± 3.7 mm). In addition, the braided cords produced the lowest TNF-α levels (0.43 ± 0.08pg/mL). Astringent systems such as ferric sulfate had higher toxicity in HGFs.

Conditional Mitigation of Dental-Composite Material-Induced Cytotoxicity by Increasing the Cure Time

Light-cured composite resins are widely used in dental restorations to fill cavities and fabricate temporary crowns. After curing, the residual monomer is a known to be cytotoxic, but increasing the curing time should improve biocompatibility. However, a biologically optimized cure time has not been determined through systematic experimentation. The objective of this study was to examine the behavior and function of human gingival fibroblasts cultured with flowable and bulk-fill composites cured for different periods of time, while considering the physical location of the cells with regard to the materials. Biological effects were separately evaluated for cells in direct contact with, and in close proximity to, the two composite materials. Curing time varied from the recommended 20 s to 40, 60, and 80 s. Pre-cured, milled-acrylic resin was used as a control. No cell survived and attached to or around the flowable composite, regardless of curing time. Some cells survived and attached close to (but not on) the bulk-fill composite, with survival increasing with a longer curing time, albeit to <20% of the numbers growing on milled acrylic even after 80 s of curing. A few cells (<5% of milled acrylic) survived and attached around the flowable composite after removal of the surface layer, but attachment was not cure-time dependent. Removing the surface layer increased cell survival and attachment around the bulk-fill composite after a 20-s cure, but survival was reduced after an 80-s cure. Dental-composite materials are lethal to contacting fibroblasts, regardless of curing time. However, longer curing times mitigated material cytotoxicity exclusively for bulk-fill composites when the cells were not in direct contact. Removing the surface layer slightly improved biocompatibility for cells in proximity to the materials, but not in proportion to cure time. In conclusion, mitigating the cytotoxicity of composite materials by increasing cure time is conditional on the physical location of cells, the type of material, and the finish of the surface layer. This study provides valuable information for clinical decision making and novel insights into the polymerization behavior of composite materials.

Long‐term exposure to cigarette smoke influences characteristics in human gingival fibroblasts

Periodontal disease is a chronic inflammatory disease caused by periodontopathic bacteria accumulated in the gingival sulcus and periodontal pocket. Cigarette smoking is a well-established risk factor for periodontal disease, and periodontal tissues in smokers are chronically exposed to cigarette smoke on a long-term basis. In this study, we investigated the effects of long-term exposure to nicotine or cigarette smoke condensate (CSC) on cellular functions of human gingival fibroblasts (HGFs). In vitro-maintained HGFs were divided into two groups. The HGFs of the short-term and the long-term culture groups were cultured for 4 and 25days, respectively, in the presence or absence of nicotine, which is one of the main components of cigarette smoke, or CSC. The cellular proliferation and migration capacities of HGFs exposed to nicotine or CSC were evaluated by WST-1 and wound healing assays. The effects of exposure to nicotine or CSC on the expression of various extracellular matrix (ECM) components, inflammatory cytokines, and senescence-related genes were examined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The cellular senescence of HGFs exposed to nicotine or CSC was detected by the senescence-associated β-galactosidase (SA-β-gal) assay. To explore the senescence-associated microRNA (miRNA), we extracted miRNA from the HGFs and the expression profiles were examined by miRNA array. In short-term culture, no significant changes were observed. Long-term exposure of HGFs to nicotine or CSC significantly suppressed their cellular proliferation and migration and upregulated type Ⅰ collagen, type Ⅲ collagen, interleukin (IL)-6, IL-8, p16, p21, and p53mRNA expression, and IL-6 and IL-8 protein expression. Furthermore, long-term nicotine or CSC exposure significantly increased the percentage of SA-β-gal-positive HGFs. In addition, long-term nicotine or CSC exposure reduced miR-29b and miR-199a expression to less than 50% of that in the unstimulated HGFs. These data suggest that long-term smoking habits may reduce wound healing ability, modulate ECM protein homeostasis, stimulate the inflammatory response, and accelerate cellular senescence in HGFs, and consequently accelerate the progression of periodontal diseases.

Aromatase inhibitor anastrozole modifies cellular functions in gingival fibroblasts and endothelial cells: possible periodontal complications of aromatase inhibitor treatment.

Recent studies have shown that treatment with aromatase inhibitors contributes to an increased prevalence of periodontitis. In this study, we assessed effects of the aromatase inhibitor anastrozole on cellular function of human gingival fibroblasts (HGFs) and endothelial cells. Expression levels of collagen, extracellular matrix (ECM) proteins, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) were examined in HGFs exposed to anastrozole. Furthermore, inflammatory responses in HGFs cultured with anastrozole were evaluated in the presence of Porphyromonas gingivalis lipopolysaccharide. We also evaluated the vascular permeability and vascular endothelial (VE)-cadherin expression of endothelial cells exposed to anastrozole. Anastrozole enhanced expression levels of collagen, ECM proteins, TIMPs, and inflammatory cytokines in HGFs, as well as vascular permeability of endothelial cells. In addition, anastrozole reduced expression levels of MMPs in HGFs and VE-cadherin in endothelial cells. These results suggest that anastrozole modulates various cellular functions in HGFs and endothelial cells.

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.

In Vitro Induction of Epithelialization of Human Gingival Fibroblasts by Mesenchymal Stem Cells Conditioned Medium (MSCCM) and Its Effects on the Secretion of Interleukin-6 and Interleukin-1beta

Epithelial injury is difficult to restore normal structure and function upon self-repair. Tissue engineering technology provides hope for epithelial injury. It is showed that human gingival fibroblasts (HGFs) could be ideal seed cells for tissue repair. Our study intends to assess mesenchymal stem cell (MSC) conditioned medium’s effect on HGFs epithelization. HGFs were cultured In Vitro and divided into two groups, including control maintained at complete DMEM medium containing 10% FBS and induction group treated by MSC conditioned medium followed by analysis of cell proliferation by MTT assay, cell apoptosis, Keratin and vimentin expressions by real-time PCR and Western blot. IL-6 and IL-1β secretions by ELISA. MSC conditioned medium induction significantly inhibited HGFs proliferation, increased keratin mRNA and protein levels, reduced vimentin expression, and enhanced IL-6 and IL-1β secretions compared with control (P &lt; 0.05). Caspase 3 activity failed to exhibit statistical significance (P &gt; 0.05). MSC conditioned medium induced HGFs epithelization and promoted IL-6 and IL-1β secretions, thus providing theoretical basis for the repair function of HGFs.

The responses of human gingival fibroblasts to magnesium-doped titanium.

The titanium (Ti) implant is widely used in implant dentistry; yet peri-implantitis has always been one of the most common and serious complications. Here, we demonstrated that magnesium-doping would be an effective way of enhancing the integration between implant surfaces and gingival tissues, which is critical to peri-implant health. The magnesium (2.76-6.35 at %) was immobilized onto the titanium substrate by a magnesium plasma immersion ion implantation (Mg-PIII) technique. Mg-PIII treatments did not alter surface topographies of the original titanium substrate but improved its hydrophilicity. The in vitro study including cell viability, adhesion, proliferation, migration, and real-time polymerase chain reaction assays disclosed improved adhesion, proliferation, migration, and extracellular matrix remodeling abilities of human gingival fibroblasts (HGFs) on the magnesium-doped titanium. The results of western blot suggested that the Mg-modified titanium induced the phosphorylation of AKT through the activation of PI3K. Our results revealed that magnesium-doping would potentially enhance soft tissue sealings by promoting cellular functions of HGFs in a dose-dependent manner, boding well for its applications on surfaces of implant necks in early peri-implant soft tissue integrations.

LncRNA MALAT1 regulates inflammatory cytokine production in lipopolysaccharide-stimulated human gingival fibroblasts through sponging miR-20a and activating TLR4 pathway.

It has been reported that long non-coding RNAs (lncRNAs), such as metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), act as key regulators of the development of inflammatory diseases. However, it is unclear whether MALAT1 regulates the function of human gingival fibroblasts (HGFs) in periodontitis. This study is to explore the role of MALAT1 on inflammatory cytokine production of HGFs. Primary HGFs were harvested from human gingiva. MALAT1 was detected in inflammatory and healthy gingival tissues via quantitative real-time PCR (qRT-PCR). Bioinformatics analysis, dual-luciferase reporter assay, and RNA-binding protein immunoprecipitation (RIP) were used to detect the relationship among MALAT1, toll-like receptor 4 (TLR4), and microRNA (miR) -20a. After transfection LPS-treated HGFs with MALAT1 siRNA (si-MALAT1), miR-20a mimic or overexpression MALAT1 plasmid (sno-MALAT1), the levels of MALAT1, miR-20a, TLR4, IL-6 and IL-8 were analyzed by qRT-PCR, enzyme-linked immunosorbent assay, or western blot assay. MALAT1 up-regulated in inflammatory gingival tissues of chronic periodontitis. MiR-20a was bound with MALAT1 and TLR4 3'-UTR in RNA-protein complex with Ago2, respectively. Moreover, MALAT1, TLR4, IL-6, and IL-8 increased while miR-20a decreased after 1μg/mL Porphyromonas gingivalis lipopolysaccharide (LPS) or Escherichia coli LPS stimulation. MiR-20a inhibited the expression of proinflammatory cytokines via binding to TLR4 3'-UTR. In addition, MALAT1 increased TLR4 level and the secretion of inflammatory cytokines. MALAT1 enhances inflammatory cytokine production through sponging miR-20a and releasing TLR4, indicating a regulatory role of MALAT1 in periodontal inflammation.

Effects of surface roughness of Ce‐TZP Al2O3 on the morphology and function of human gingival fibroblasts

Background While the function of dentogingival junction, the epithelial and connective tissue attachments to the tooth, has been well documented, the attachment of peri-implant soft tissues to dental implants remains uncertain. Recently, dental implants made of zirconia (Zr) have been developed. Our previous study demonstrated the strength of osseointegration of Ce-TZP Al2O3 was greater than that of acid etched Ti in a rat model. However, the attachment of Ce-TZP Al2O3 to the soft-tissues remains unknown. Aim/Hypothesis In order to evaluate the capability of Ce-TZP Al2O3 to achieve the soft tissue attachment, this study aimed to assess the attachment, proliferation, morphology, and extracellular matrix (ECM) synthesis of HGF-1 on Ce-TZP Al2O3 and Ti surfaces with different grades of roughness. Material and Methods We prepared three types of Ce-TZP Al2O3 substrates with different grades of surface roughness (mirror, smooth and roughZr) as test surfaces, and two types of Ti (machined and mirrorTi) substrates as control. We observed the surface characteristics of all samples, including topography, elemental composition, and hydrophilicity using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and contact angle goniometer, respectively. Initial cell attachment and proliferative activities were evaluated after 3 hours, 24 hours, and 3 days of cultivation on the discs. Cell morphologies were observed using immunofluorescent staining with rhodamine phalloidin for actin cytoskeleton and with DAPI for the nucleus after 3 hours of incubation. Using Sirius red staining and colorimetry, we measured collagen synthesis on the HGF-1 disc cultures after 28 days. Expression of inflammatory cytokines, including TNF, were assessed on 30 minutes in the HGF-1 cultures using a cytokine ELISA systems. Results SEM images of machined Ti revealed a typical topography with wavy microgrooves. Additionally, the uniform linear structures along the polishing appeared on both rough and smooth Zr. Early response of HGF-1 fibroblasts on mirror Zr showed significantly greater than that on Ti surfaces. The highest proliferative capacities were observed on the mirror Zr compared with the other discs. Confocal microscopic images of HGF-1 staining revealed that fibroblasts on two types of Ti showed typical spindle-shapes, whereas those on the Zr discs had small round cells including contractile ring-like structures and developed cytoskeletons. HGF-1 on the mirror Zr surface was clearly larger than that on the other two Zr surfaces. The amount of collagen deposited on mirror Zr increased significantly compared with the other discs. Inflammatory cytokine production was significantly lower on mirror Zr than that on machined Ti and mirror Ti. There were no significant difference among three Zr surfaces. Conclusions and Clinical Implications The HGF-1 on mirror Zr surfaces exhibited more matured morphology and greater cellular viability and differentiation than those of other surfaces, which might be through their grater biocompatibility as demonstrated by the lower proinflammatory cytokine synthesis. These results indicate that the mirrored Ce-TZP Al2O3 surfaces may show a higher affinity for soft tissue around dental implant than Ti and the unlevel Ce-TZP Al2O3 surfaces.

Lipopolysaccharide from Porphyromonas gingivalis promotes autophagy of human gingival fibroblasts through the PI3K/Akt/mTOR signaling pathway

AimsLipopolysaccharide (LPS) is a major component of cell wall in gram-negative bacteria and has been proved to be a predominant pathogenic factor in periodontitis. Porphyromonas gingivalis (P.g) was abundant in patients with periodontitis and was associated with patient clinic-pathological characteristics. Furthermore, autophagy is a potential mechanism in inflammatory disease. In this study, we hypothesized that LPS from P.g may affect the physiological functions of human gingival fibroblasts (HGFs) through activating cellular autophagy. However, it remains unclear what molecular basis related to LPS-induced autophagy in HGFs. Main methodsHere, we initially addressed the contribution of LPS from P.g in inducing autophagy in HGFs. Through a combination of morphology and quantification approaches involving autophagosomes formation observation as well as microtubule-associated protein light chain 3 (LC3)-II conversion. We further evaluated whether the PI3K/Akt/mTOR signaling could mediate LPS-induced autophagy in HGFs. Key findingsOur results revealed that autophagy was more obvious in LPS-treated cells compared with that in control groups. Finally, our results demonstrated that LPS from P.g promoted autophagy in HGFs and was negatively regulated by PI3K/Akt/mTOR. SignificanceAnalysis of these data implicates that LPS from P.g has a significant impact on the autophagy of HGFs by suppressing PI3K/Akt/mTOR signaling pathway.

Ultraviolet light-treated zirconia with different roughness affects function of human gingival fibroblasts in vitro: the potential surface modification developed from implant to abutment.

To evaluate whether ultraviolet (UV) light irradiation of smooth and rough zirconia disks enhances its biocompatibility with human gingival fibroblasts (HGFs). Zirconia disks were divided into four groups: smooth control (S-C), smooth with UV light treatment (S-UV), rough control (R-C), and rough with UV light treatment (R-UV). The surface morphology and wettability were analyzed, and X-ray photoelectron spectroscopy was carried out. The cultured HGFs' adhesive density, morphology, proliferation, and collagen synthesis were measured on different time points from 3 h to 7 days. After 24-h UV light treatment, contact angles decreased from 51.98° (S-C) and 63.87° (R-C) to 33.76° (S-UV) and 36.15° (R-UV). X-ray photoelectron spectroscopy analysis showed that surface C/O ratio reduced from 1.86 and 2.39 to 1.33 and 1.19. After UV light treatment, cells initial spreading areas and perimeters were doubled on S-UV disks and nearly tripled on R-UV disks. Three hours cell adhesion was enhanced on S-UV disks, and 24-h cell density was increased on R-UV. Cell proliferation of 48 or 72 h was all significantly changed on UV-treated disks. Cells on R-UV also released highest level of Col-1 after 3 and 7 days, whereas those on S-UV produced less levels compared with control. Our study demonstrated for the first time that UV light treatment on rough (Ra = 0.19 ± 0.03 μm) zirconia had a positive effect on behavior of HGFs, including 24-h cell adhesion, proliferation, and collagen release. The effect varied with surface roughness and time point. Surface morphology and hydrophilicity, as two interacted factors, both are governed on HGFs behavior.

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.

Protein kinase-A-dependent osteoprotegerin production on interleukin-1 stimulation in human gingival fibroblasts is distinct from periodontal ligament fibroblasts

Periodontitis, a chronic inflammatory disease, is characterized by increased expression of interleukin (IL)-1 and other inflammatory mediators resulting in extensive osteoclast formation and bone loss. Expression of receptor activator of nuclear factor kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), by osteoblasts is important to regulate osteoclast differentiation. The aim of the present study was to investigate the regulatory effects of IL-1 on RANKL and OPG production by mesenchymal fibroblasts in periodontal tissue. Human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (PDL) were stimulated with IL-1alpha with or without protein synthesis inhibitor cycloheximide (CHX), protein kinase A (PKA) inhibitors, protein kinase C (PKC) inhibitors and prostaglandin E(2) (PGE(2)) inhibitor. In some experiments, the cultured cells were directly stimulated with either PKA or PKC activators. In HGF, IL-1alpha-stimulated OPG mRNA expression was high and could be reduced by CHX. PKA inhibitor completely abrogated IL-1alpha-induced OPG mRNA expression and OPG production. Endogenous PGE(2) further enhanced IL-1alpha-induced OPG production in HGF. In PDL, RANKL mRNA expression was greatly augmented by IL-1alpha. IL-1alpha induced OPG mRNA expression and protein production. PKC inhibitor partially reduced IL-1alpha-induced OPG production and PKC activator enhanced OPG production in PDL. The IL-1alpha-stimulated OPG mRNA expression in HGF was greater than PDL. These results provide new evidence for the possible osteoclastogenesis-inhibitory function of HGF through PKA activity pathway. PDL utilized PKC for OPG production. Thus, we emphasize that HGF and PDL have different characteristics of host defence mechanism against inflammatory process.

Outer Membrane Vesicles From Porphyromonas gingivalis Affect the Growth and Function of Cultured Human Gingival Fibroblasts and Umbilical Vein Endothelial Cells

The purpose of this study was to examine the effects of outer membrane vesicles (OMV) obtained from Porphyromonas gingivalis (Pg) on the growth and function of human gingival fibroblasts (HGF) and human umbilical vein endothelial cells (HUVEC). OMV were obtained from a cell-free growth medium of Pg ATCC 33277 by 40% NH2SO4 precipitation and ultracentrifugation. Cell proliferation was measured by 3H-thymidine incorporation into growing HGF and HUVEC. Endothelial cell function was determined by their capacity to form a network of capillary tubes on an extracellular matrix (ECM). Proliferating HGF and HUVEC demonstrated a significant dose-dependent inhibition of 3H-thymidine uptake when cultured with 0 to 40 microg/ml of OMV protein. HGF and HUVEC showed an IC50 of growth of about 9.0 microg/ml and 4.5 microg/ml of OMV protein, respectively. Capillary tube formation by HUVEC cultured on an ECM was suppressed by 70% to 80% with 5 microg/ml OMV protein after 18 hours of incubation. The presence of proteolytic enzymes in the OMV did not contribute to capillary tube disruption, since blocking enzyme activity with specific inhibitors did not reduce the suppression of capillary tube formation. After heating at 90 degrees C for 5 minutes, OMV significantly lost their capacity to suppress capillary tube formation. OMV significantly inhibit the proliferation of cultured HGF and HUVEC in a dose-dependent manner. OMV suppressed the capillary tube formation by cultured HUVEC. The factor(s) appeared to be a protein and not endotoxin because its inhibitory activity was markedly reduced by heat inactivation. These studies suggest that OMV contribute to chronic periodontitis by suppressing cell proliferation and revascularization in periodontal tissues.

Adenosine regulates the IL-1 beta-induced cellular functions of human gingival fibroblasts.

In this study we examined the influence of adenosine on the cellular functions of human gingival fibroblasts (HGF), such as the production of inflammatory cytokines and extracellular matrices (ECM), and the expression and function of adhesion molecules. Concerning the expression of adenosine receptors, RT-PCR analysis revealed that HGF expressed adenosine receptor A1, A2a and A2b, but not A3 mRNA. Ligation of adenosine receptors by adenosine or its related analogue, 2-chloroadenosine (2-CADO), N(6)-cyclopentyladenosine (CPA) or CGS21680 synergistically increased IL-1beta-induced IL-6 and IL-8 production. In terms of ECM expression, adenosine and the adenosine receptor agonists, 2-CADO and CPA, enhanced constitutive and IL-1beta-induced expression of hyaluronate synthase mRNA, but not the mRNA levels of other ECM, such as collagen type I, III and fibronectin. Moreover, the adherence of IL-1beta-stimulated HGF to activated lymphocytes was also inhibited by adenosine, which is in part explained by the fact that adenosine down-regulated the IL-1beta-induced expression of ICAM-1 on HGF. These results provide new evidence for the possible involvement of adenosine in the regulation of inflammatory responses in periodontal tissues.

Impairment of gingival fibroblast adherence by IL-6/sIL-6R.

Interleukin-6 (IL-6) binds to human gingival fibroblasts (HGF) in the presence of a soluble form of IL-6 receptor (sIL-6R). We investigated the effects of IL-6 on the functions of HGF in the presence of sIL-6R. HGF changed their morphology from spindle-shaped to round, and detached from the culture dish by stimulation with IL-6/sIL-6R. In this condition, a signal transducer gp130 and a transcription factor Stat3 were phosphorylated, resulting in activation of transcription factors Stat3 and C/EBPbeta. Cytoskeletal beta-actin and adhesion molecule integrin-alpha5, a subunit of alpha5beta1 integrin (VLA-5), were found to possess potential binding domains for these transcription factors in their promoters. Accumulation of beta-actin and integrin-alpha5 mRNA decreased, contrary to the expectation of the induction of gene transcription. Furthermore, the decrease in their mRNAs was associated with reduced expression of both actin and VLA-5 proteins. These results suggest that the expression of VLA-5 and actin was down-regulated in HGF through an IL-6 signaling pathway, resulting in impairment of HGF adherence.

Microtubules and vimentin associated filaments (VIFs) in cultured human gingival fibroblasts (HGFs) after exposure to acrolein and acetaldehyde

Tobacco smoke, particularly its non-volatile fraction e. g. nicotine, is considered to be a major risk factor for the development and progression of periodontal disease. The purpose of this study has been to determine the effects of acrolein and acetaldehyde of the volatile fraction of tobacco smoking, on human gingival fibroblasts (HGFs) cultured in vitro with particular attention to cytoskeletal structures. A human gingival fibroblast strain derived from healthy gingiva was utilized in this study. The cells were exposed to acrolein and acetaldehyde at various concentrations. Control and treated cells were compared as regards their adhesion on cell culture dishes. Their cytoskeletal structures [tubulin and vimentin intermediate filaments (VIFs)] were examined by fluorescence microscopy. The results revealed that both substances produced similar effects resulting in a dose dependent decrease in cell adhesion and alterations of HGF cytoskeleton consisting of rearrangement and/or disruption of microtubules and vimentin associated filaments. Changes in cell shape and decrease in cell size were also seen. On the basis of this in vitro study, it appears that tobacco, through its volatile components, may directly affect the main functions of HGFs.