Influence of titanium and zirconia substrates on the synthesis of inflammatory mediators.

The repair and homeostasis of peri-implant tissues depend on several factors such as the local presence of pathogenic bacteria and their products. Among other events, peri-implant tissue response is also related to the implant material used, which interferes with cells and extracellular matrix interactions, affecting the osseointegration process. In this study, the influence of zirconia (Zr) and titanium (Ti) substrates on the response of preosteoblasts (MC3T3) and murine macrophages (RAW 264.7) exposed to lipopolysaccharide (LPS, P. gingivalis) was evaluated. Zr and Ti disks were obtained and subjected to surface roughness standardization, which was analyzed by scanning electronic microscopy (SEM). The cells were subsequently cultured on Zr and Ti surfaces in AlphaMEM culture medium for 24 h, followed by LPS stimulus for 4 h. The production of reactive oxygen species (ROS) and gene expression of inflammatory markers were determined. SEM images showed that Ti disks exhibited higher surface roughness than that of Zr disks. Cells that seeded onto Ti and Zr had increased expression of inflammatory mediators and ROS production in the presence of LPS; however, such cell responses were more evident for Ti disks. These data indicate that contact of cells with Zr surfaces may lead to a lower inflammatory potential than Ti surfaces. Elucidation of the inflammatory response triggered by LPS for cells in contact with titanium and zirconia may contribute to the selection of materials for installation of osseointegrated implants.

EGF coating of titanium surfaces modulates cytokines in oral mucosal primary cells exposed to TNF-α.

OBJECTIVE: This study assessed the metabolism of oral mucosal cells cultured on titanium discs (Ti) coated (or not) with epidermal growth factor (EGF) and exposed to tumor necrosis factor alpha (TNF-α). METHODS: Fibroblasts or keratinocytes were seeded on Ti coated or not with EGF, and then exposed to 100 ng/mL of TNF-α for 24 h. Groups were established: G1: Ti (control); G2: Ti + TNF-α; G3: Ti + EGF; and G4: Ti + EGF + TNF-α. Both cell lines were evaluated for: viability (AlamarBlue®, n = 8); interleukin 6 and 8 (IL-6, IL-8) gene expression (qPCR, n = 5), and protein synthesis (ELISA, n = 6). For keratinocytes cells, the matrix metalloproteinase type 3 (MMP-3) was evaluated by qPCR (n = 5) and ELISA (n = 6). A 3-D culture of fibroblasts was analyzed by confocal microscopy. The data were subjected to ANOVA analysis, α = 5%. RESULTS: Increased cell viability was observed in all groups compared with G1. Enhanced gene expression and synthesis of IL-6 and IL-8 by fibroblasts and keratinocytes in G2 and modulation of hIL-6 gene expression in G4 was noted. Modulation of IL-8 synthesis occurred in keratinocytes in G3 and G4. Keratinocytes in G2 showed enhanced gene expression of hMMP-3. A 3-D culture showed more cells in G3. Fibroblasts in G2 exhibited disrupted cytoplasmic membrane. Cells in G4 showed elongated morphology with intact cytoplasm. CONCLUSIONS: EGF coating increases cell viability and modulates the response of oral cells exposed to an inflammatory stimulus.

Naringenin and proanthocyanidins pre-treatment decreases synthesis and activity of gelatinases induced by zoledronic acid in a dental implant surface in vitro model.

OBJECTIVE: To assess the effects of pre-treatment with proanthocyanidins (PA) flavonoids, from grape seed extract, and synthetic naringenin (NA) on the synthesis of matrix metalloproteinases (MMPs) gelatinases and their tissue inhibitors (TIMPs), as well as the gelatinolytic activity of MMPs by human gingival fibroblasts (HGF) and osteoblasts (Ob) exposed to zoledronic acid (ZA) in a dental implant surface in vitro model. DESIGN: The highest non-cytotoxic concentrations of NA and PA were determined for HGF (10 µg/mL; defined by previous study) and Ob (0.5 µg/mL; defined by prestoBlue assay). Then, HFG and Ob were individually seeded onto titanium discs, and after 24 h, cells were pre-treated (or not) with NA or PA, followed (or not) by exposure to ZA. Next, MMP-2, MMP-9, TIMP-1, TIMP-2 synthesis (ELISA), and gelatinolytic activity (in situ zymography) was evaluated. Data were analyzed by one-way ANOVA and Tukey tests (α = 0.05). RESULTS: ZA treatment increased the synthesis (p < 0.05) and activity of MMPs; flavonoids pre-treatment controlled ZA-induced gelatinolytic effects, down-regulating MMPs synthesis (p < 0.05) and activity by HGF and Ob. For HGF, NA and PA pre-treatment did not up-regulate TIMP synthesis after ZA exposure (p > 0.05); for Ob, TIMP-2 was up-regulated (p < 0.05) by flavonoids, followed by ZA. CONCLUSIONS: NA and PA pre-treatment provides interesting results in the modulation of ZA deleterious effects, down-regulating MMP-2 and MMP-9 synthesis and activity by HGF and Ob and up-regulating TIMP-2 by Ob.

Titanium alkalinization improves response of osteoblasts to zoledronic acid.

This investigation is aimed to determine the effect of the modification of titanium surface with NaOH on the metabolism of osteoblasts treated with zoledronic acid (ZA). Machined and NaOH-treated titanium disks were used. Surfaces were characterized by scanning electron microscopy, confocal microscopy, and x-ray photoelectron spectroscopy (XPS) analysis. Human osteoblasts were seeded onto the disks. After 24 h, cells were treated with ZA at 5 µM for 7 days. At this point, cell viability, collagen synthesis, total protein production, alkaline phosphatase activity, and mineral nodule deposition were assessed. The results of surface roughness were descriptively and statistically analyzed (t-Student), while the XPS results were qualitatively described. Cell metabolism data were analyzed by the analysis of variance two-way and Tukey tests at a 5% significance level. The results demonstrated that NaOH-treatment increased surface roughness (p < .05) and confirmed the presence of sodium titanate and a pH switch on the NaOH-treated disks. This modification also resulted in higher cell viability, collagen synthesis, total protein production, and alkaline phosphatase by osteoblasts when compared to cells seeded onto machined disks (p < 0.05). In the presence of ZA, all cellular metabolism and differentiation parameters were significantly reduced for cells seeded on both surfaces (p < 0.05); however, the cells seeded onto modified surfaces showed higher values for these parameters, except for mineral nodule deposition (p < 0.05). NaOH modification improved cell adhesion and metabolism of osteogenic cells even in the presence of ZA. The surface modification of titanium with NaOH solution may be an interesting strategy to improve metabolism and differentiation of osteoblasts and accelerate osseointegration process, mainly for tissues exposed to ZA.

Regulation of interleukin-6 and matrix metalloproteinases syntheses by bioflavonoids and photobiomodulation in human gingival fibroblasts.

This study aimed to evaluate the separately effects of bioflavonoids proanthocyanidins, from grape seed extract (GSE) and synthetic naringenin (NA), as well as photobiomodulation (PBM) by low-level laser therapy on interleukin (IL)-6 and matrix metalloproteinases (MMPs) syntheses by human gingival fibroblasts (HGF). For this purpose, a connective tissue exposure (ulceration) model of HGF, stimulated with tumor necrosis factor-alpha (TNF-α), was used. Initially, the highest non-cytotoxic and non-genotoxic concentrations of bioflavonoids were determined by cell viability and micronuclei formation assays. Then, HGF were exposed to different stimuli: culture medium (negative control), dimethyl sulfoxide (DMSO), TNF-α, NA, GSE, TNF-α + NA, TNF-α + GSE, PBM (3 J/cm2, 0.025 W, 780 nm), and TNF-α + PBM. Next, IL-6, MMP-2, and MMP-9 syntheses were assessed. The concentration of 10 µg/mL of bioflavonoids increased cell viability at 24 and 48 h and did not present cytotoxic or genotoxic effects on HGF after 24, 48, and 72 h of contact. This concentration was selected for the assessment of bioflavonoids potential in modulating inflammatory mediators. TNF-α exposure enhanced IL-6 (170%), MMP-2 (10%), and MMP-9 (20%) syntheses, while a decrease of MMP-2 by 55% after exposure to TNF-α + GSE and 20% after TNF-α + NA and TNF-α + PBM was observed. MMP-9 synthesis was decreased by 35% after TNF-α + NA, 20% after TNF-α + GSE, and 30% after PBM. IL-6 was down-regulated by GSE in the presence of TNF-α (80%). In conclusion, TNF-α up-regulated IL-6 and MMPs, while bioflavonoids and PBM down-regulated MMP-2 and MMP-9 syntheses; GSE also decreased IL-6 synthesis, demonstrating the individual promising potential of these therapies for ulceration management.

Photobiomodulation using LLLT and LED of cells involved in osseointegration and peri-implant soft tissue healing.

This study evaluated the influence of photobiomodulation (PBM) using low-level laser therapy (PBM/LLLT) or light-emitting diode (PBM/LED) therapy on peri-implant tissue healing. A laboratory model was used to assess the adhesion and metabolism of osteoblasts (SaOs-2), human gingival fibroblasts (HGF), and normal oral keratinocytes (NOK) seeded on a titanium (Ti) surface. After seeding the cells on disks of Ti placed in wells of 24-well plates, three irradiations were performed every 24 h at energy density of 3 J/cm2. For PBM/LLLT, a LaserTABLE device was used with a wavelength of 780 nm and 25 mW, while for PBM/LED irradiation, a LEDTABLE device was used at 810 nm, 20 mW, at a density of 3 J/cm2. After irradiations, the number of cells (NC) attached and spread on the Ti surface, cell viability (CV), total protein (TP), and collagen (Col) synthesis were assessed. Alkaline phosphate activity (ALP) was evaluated only for SaOs-2. Data were submitted to ANOVA complemented by Turkey statistical tests at a 5% significance level. PBM significantly increased adherence of NOK to the Ti surface, while no significant effect was observed for SaOs-2 and HGF. PBM positively affected CV, as well as Col and TP synthesis, in distinct patterns according to the cell line. Increased ALP activity was observed only in those cells exposed to PBM/LLLT. Considering cell specificity, this investigation reports that photobiomodulation with low-power laser and LED at determined parameters enhances cellular functions related to peri-implant tissue healing in a laboratory model.

Extracellular-Vesicle-Based Coatings Enhance Bioactivity of Titanium Implants-SurfEV.

Extracellular vesicles (EVs) are nanoparticles released by cells that contain a multitude of biomolecules, which act synergistically to signal multiple cell types. EVs are ideal candidates for promoting tissue growth and regeneration. The tissue regenerative potential of EVs raises the tantalizing possibility that immobilizing EVs on implant surfaces could potentially generate highly bioactive and cell-instructive surfaces that would enhance implant integration into the body. Such surfaces could address a critical limitation of current implants, which do not promote bone tissue formation or bond bone. Here, we developed bioactive titanium surface coatings (SurfEV) using two types of EVs: secreted by decidual mesenchymal stem cells (DEVs) and isolated from fermented papaya fluid (PEVs). For each EV type, we determined the size, morphology, and molecular composition. High concentrations of DEVs enhanced cell proliferation, wound closure, and migration distance of osteoblasts. In contrast, the cell proliferation and wound closure decreased with increasing concentration of PEVs. DEVs enhanced Ca/P deposition on the titanium surface, which suggests improvement in bone bonding ability of the implant (i.e., osteointegration). EVs also increased production of Ca and P by osteoblasts and promoted the deposition of mineral phase, which suggests EVs play key roles in cell mineralization. We also found that DEVs stimulated the secretion of secondary EVs observed by the presence of protruding structures on the cell membrane. We concluded that, by functionalizing implant surfaces with specialized EVs, we will be able to enhance implant osteointegration by improving hydroxyapatite formation directly at the surface and potentially circumvent aseptic loosening of implants.

New Multiscale Characterization Methodology for Effective Determination of Isolation-Structure-Function Relationship of Extracellular Vesicles.

Extracellular vesicles (EVs) have been lauded as next-generation medicines, but very few EV-based therapeutics have progressed to clinical use. Limited clinical translation is largely due to technical barriers that hamper our ability to mass produce EVs, i.e., to isolate, purify, and characterize them effectively. Technical limitations in comprehensive characterization of EVs lead to unpredicted biological effects of EVs. Here, using a range of optical and non-optical techniques, we showed that the differences in molecular composition of EVs isolated using two isolation methods correlated with the differences in their biological function. Our results demonstrated that the isolation method determines the composition of isolated EVs at single and sub-population levels. Besides the composition, we measured for the first time the dry mass and predicted sedimentation of EVs. These parameters were likely to contribute to the biological and functional effects of EVs on single cell and cell cultures. We anticipate that our new multiscale characterization approach, which goes beyond traditional experimental methodology, will support fundamental understanding of EVs as well as elucidate the functional effects of EVs in in vitro and in vivo studies. Our findings and methodology will be pivotal for developing optimal isolation methods and establishing EVs as mainstream therapeutics and diagnostics. This innovative approach is applicable to a wide range of sectors including biopharma and biotechnology as well as to regulatory agencies.

Influence of bisphosphonates on oral implantology: Sodium alendronate and zoledronic acid enhance the synthesis and activity of matrix metalloproteinases by gingival fibroblasts seeded on titanium.

OBJECTIVE: This study aimed to assess the influence of the bisphosphonates zoledronic acid and sodium alendronate on MMP-2 and MMP-9 synthesis and activity by gingival fibroblasts seeded onto titanium substrate. DESIGN: Titanium discs were placed in 24-well cell culture plates and gingival fibroblasts were seeded (1 × 105 cells/discs) on them using Dulbecco's Modified Eagle's Medium (DMEM) + 10 % fetal bovine serum (FBS) for 24 h. After this period, a fresh serum-free DMEM containing zoledronic acid or sodium alendronate at 0.5 µM, 1 µM or 5 µM was applied on the cells for an additional of 24 h. Serum-free DMEM and tumor necrosis factor alpha (TNF-α) were used as negative and positive controls, respectively. MMP-2 and MMP-9 synthesis and activity were determined by ELISA (Enzyme-Linked Immunosorbent Assay) and conventional/in situ zymography. Quantitative data were analyzed by one-way ANOVA and Tukey's tests (α = 0.05). The in situ zymography data were qualitatively described. RESULTS: Despite both bisphosphonates increased the MMPs synthesis, this effect was significant higher in zoledronic acid groups. MMPs activity resembled by gelatinolytic activity was also enhanced by sodium alendronate and zoledronic acid in a similar pattern. CONCLUSIONS: Zoledronic acid and sodium alendronate increased in a dose-dependent manner MMP-2 and MMP-9 synthesis by gingival fibroblasts seeded on titanium. MMP-2 activity was up-regulated by zoledronic acid treatment.

Chemotherapy drugs and inflammatory cytokines enhance matrix metalloproteinases expression by oral mucosa cells.

OBJECTIVE: Oral mucositis (OM), the most common side effect of cancer therapy, is associated with pro-inflammatory cytokines and matrix metalloproteinases (MMPs) increased expression. Although there are approaches for OM management, none is infallible, thus, elucidation of molecular events related to OM etiopathogenesis may improve current therapeutic strategies. This study assessed the influence of pro-inflammatory cytokines and chemotherapy drugs on MMPs expression and synthesis by oral mucosa cells. DESIGN: Human gingival fibroblasts (HGF) were exposed to different concentrations of methotrexate (MTX) and 5-fluorouracil (5-FU); subsequentially, cell viability, nitric oxide and interleukin(IL)-6 production were evaluated to select the concentration of these drugs that could stimulate inflammatory phenotype without cytotoxic effects. Then, HGF and primary gingival keratinocytes (PGK) were subjected to different stimuli: culture medium (negative control), tumor necrosis factor-alpha (TNF-α - positive control), IL-6, IL-8, MTX, and 5-FU for 3, 6, 12, and 24 h. Next, gene expression and synthesis of MMP-2 and MMP-9 by HGF and MMP-3 by PGK were assessed. RESULTS: At 6 h, MMP-2 synthesis increased 60 % after exposure to TNF-α and MTX, 40 % after IL-6, and 15 % after IL-8. At 12 h, MMP-9 synthesis increased 15 % after exposure to TNF-α, while MMP-3 synthesis increased 30 % after TNF-α, and 10 % after IL-8. TNF-α-treated groups presented increased gene expression of all MMPs evaluated. IL-8 and 5-FU increased MMP-2 and MMP-3 expression, while IL-6 and MTX augmented MMP-2 expression. CONCLUSIONS: The chemotherapy drugs and cytokines investigated up-regulated MMPs expression by oral mucosa cells, which may lead to OM establishment and severity.

Effects of EGF-coated titanium surfaces on adhesion and metabolism of bisphosphonate-treated human keratinocytes and gingival fibroblasts.

OBJECTIVE: To assess the effects of epidermal growth factor (EGF)-coated titanium (Ti) discs on the adhesion and metabolism of keratinocytes and gingival fibroblasts exposed to nitrogen-containing bisphosphonates. MATERIALS AND METHODS: Keratinocytes and fibroblasts were seeded (1 × 105 cells/disc) on Ti discs coated with EGF (100 nM). After 24 h, cells were exposed or not to sodium alendronate (SA) or zoledronic acid (ZA) at different concentrations (0 = control, 0.5, 1, or 5 µM) for 48 h. Cell adhesion to the substrates was evaluated by fluorescence microscopy. Cell viability (alamarBlue, n = 6) and synthesis of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2), and keratinocytes growth factor (KGF) (ELISA, n = 6) were assessed. Data were statistically analyzed by one-way ANOVA and Tukey tests (α = 0.05). RESULTS: Higher cell adhesion rate was observed when keratinocytes and fibroblasts were seeded onto EGF-coated discs in comparison to uncoated discs. ZA treatment hindered the adhesion of both cell lines on the Ti discs as well as reduced the viability and synthesis of VEGF, KGF and MMP-2 by cells (p < 0.05). SA treatment did not affect cell viability, but interfered negatively on the adhesion and synthesis of EGF and KGF by the cells (p < 0.05). EGF-coated surface increased cell viability and synthesis of growth factors as well as downregulated the synthesis of MMP-2 in comparison to control (p < 0.05). CONCLUSION: EGF applied on Ti surface improves the biological responses of oral mucosa cells exposed to SA and ZA. CLINICAL RELEVANCE: EGF-coating on titanium may be a suitable strategy to improve oral mucosa cellular events related to biological sealing, especially for patients under bisphosphonate therapy.

In vitro effects of photobiomodulation applied to gingival fibroblasts cultured on titanium and zirconia surfaces and exposed to LPS from Escherichia coli.

Photobiomodulation (PBM) therapy is used to stimulate cell proliferation and metabolism, as well as reduce inflammatory cytokine synthesis, which plays a main role in the long-term stability of implants. This study assessed the response of gingival fibroblasts cultured on titanium (Ti) and zirconia (ZrO2), submitted to PBM and exposed to lipopolysaccharide (LPS). Cells seeded on Ti and ZrO2 were irradiated (InGaAsP; 780 nm, 25 mW) 3 times, using 0.5, 1.5, and 3.0 J/cm2 doses, and exposed to Escherichia coli LPS (1 µg/mL). After 24 h, cell viability (alamarBlue, n = 8), interleukin 6 (IL-6) and 8 (IL-8) synthesis (ELISA, n = 6), and IL-6 and vascular endothelial growth factor (VEGF) gene expression (qPCR, n = 5) were assessed and statistically analyzed (one-way ANOVA, α = 0.05). Cell morphology was evaluated by fluorescence microscopy. Increased cell viability occurred in all groups cultured on Ti compared with that of the control, except for cells exposed to LPS. Fibroblasts cultured on ZrO2 and LPS-exposed exhibited reduced viability. PBM at 3.0 J/cm2 and 1.5 J/cm2 downregulated the IL-6 synthesis by fibroblasts seeded on Ti and ZrO2, as well as IL-8 synthesis by cells seeded on ZrO2. Fibroblasts seeded on both surfaces and LPS-exposed showed increased IL-6 gene expression; however, this activity was downregulated when fibroblasts were irradiated at 3.0 J/cm2. Enhanced VEGF gene expression by cells seeded on Ti and laser-irradiated (3.0 J/cm2). Distinct patterns of cytoskeleton occurred in laser-irradiated cells exposed to LPS. Specific parameters of PBM can biomodulate the inflammatory response of fibroblasts seeded on Ti or ZrO2 and exposed to LPS.

Photobiomodulation of inflammatory-cytokine-related effects in a 3-D culture model with gingival fibroblasts.

The aim of this study was to assess the effects of IL-6 and IL-8 cytokines on human gingival fibroblasts (HGF) cultured in a 3-D model and the possible photobiomodulation (PBM) of such effects by low-level laser therapy. In complete culture medium (DMEM), HGF from a healthy patient were seeded in a type I collagen matrix inserted into 24-well plates. After 5 days of incubation, the cytokines were added or not to serum-free DMEM, which was applied to the cell-enriched matrices. Then, PBM was performed: three consecutive irradiations using LaserTable diode device (780 nm, 0.025 W) at 0.5 J/cm2 were delivered or not to the cells. Twenty-four hours after the last irradiation, cell viability and morphology, gene expression, and synthesis of inflammatory cytokines and growth factors were assessed. The histological evaluation demonstrated that, for all groups, matrices presented homogeneous distribution of cells with elongated morphology. However, numerous cytokine-exposed cells were rounded. IL-6 and IL-8 decreased cell viability, synthesis of VEGF, and gene expression of collagen type I. PBM enhanced cell density in the matrices and stimulated VEGF expression, even after IL-6 challenge. Reduced TNF-α synthesis occurred in those cells subjected to PBM. In conclusion, PBM can penetrate collagen matrix and stimulate HGF, highlighting the relevance of this research model for further phototherapy studies and in vitro biomodulation of the healing process.

Synergistic potential of 1α,25-dihydroxyvitamin D3 and calcium-aluminate-chitosan scaffolds with dental pulp cells.

OBJECTIVES: This study aimed to develop a porous chitosan-calcium-aluminate scaffold (CH-AlCa) in combination with a bioactive dosage of 1α,25-dihydroxyvitamin D3 (1α,25VD), to be used as a bioactive substrate capable to increase the odontogenic potential of human dental pulp cells (HDPCs). MATERIALS AND METHODS: The porous CH-AlCa was developed by the incorporation of an AlCa suspension into a CH solution under vigorous agitation, followed by phase separation at low temperature. Scaffold architecture, porosity, and calcium release were evaluated. Thereafter, the synergistic potential of CH-AlCa and 1 nM 1α,25VD, selected by a dose-response assay, for HDPCs seeded onto the materials was assessed. RESULTS: The CH-AlCa featured an organized and interconnected pore network, with increased porosity in comparison with that of plain chitosan scaffolds (CH). Increased odontoblastic phenotype expression on the human dental pulp cell (HDPC)/CH and HDPC/CH-AlCa constructs in the presence of 1 nM 1α,25VD was detected, since alkaline phosphatase activity, mineralized matrix deposition, dentin sialophosphoprotein/dentin matrix acidic phosphoprotein 1 mRNA expression, and cell migration were overstimulated. This drug featured a synergistic effect with CH-AlCa, since the highest values of cell migration and odontoblastic markers expression were observed in this experimental condition. CONCLUSIONS: The experimental CH-AlCa scaffold increases the chemotaxis and regenerative potential of HDPCs, and the addition of low-dosage 1α,25VD to this scaffold enhances the potential of these cells to express an odontoblastic phenotype. CLINICAL RELEVANCE: Chitosan scaffolds enriched with calcium-aluminate in association with low dosages of 1α,25-dihydroxyvitamin D3 provide a highly bioactive microenvironment for dental pulp cells prone to dentin regeneration, thus providing potential as a cell-free tissue engineering system for direct pulp capping.

Characterization of titanium surface coated with epidermal growth factor and its effect on human gingival fibroblasts.

OBJECTIVES: Different strategies, such as modifications on the implant abutments surface have been proposed to accelerate and improve the formation of the biological seal (BS). The aim of this study was to characterize a titanium (Ti) surface impregnated with epidermal growth factor (EGF) and to assess its influence on the metabolism and adhesion of oral mucosal cells. DESIGN: Ti discs were coated with EGF (100 nM) conjugated with a fluorophore and analyzed by fluorescence microscopy. The surface roughness analysis (Ra) of the EGF-coated Ti was performed by confocal microscopy. The EGF released in the wet environment was determined at 0, 24, 48 and 72 h by fluorimetric quantification. For assessment of the biological effects of EGF-coated Ti, gingival fibroblasts were seeded (5 × 104 cells) onto the substrate coated or not with this growth factor. After 24 h, cell adhesion and viability were evaluated by ANOVA and Tukey tests, α = .05. RESULTS: Immediate release of EGF as well as its incorporation by fibroblasts within 1 h after cells were seeded was observed. EGF-coated Ti discs presented significantly enhance surface roughness. Increased cell viability was observed on the EGF-coated discs. CONCLUSION: EGF applied to Ti discs stimulated the adhesion and metabolism of gingival fibroblasts and could be considered as an interesting alternative for improving the BS.

Photobiomodulation in the Metabolism of Lipopolysaccharides-exposed Epithelial Cells and Gingival Fibroblasts.

This study assessed the effects of photobiomodulation (PBM) to cells previously exposed to lipopolysaccharides (LPS). Human gingival fibroblasts (HGF) and epithelial cells (HaCaT) were seeded in wells of 24-well plates containing complete culture medium (DMEM). After 24 h, the DMEM was replaced by serum-free DMEM, and cells were exposed to LPS of Escherichia coli (E. coli) (10 µg mL-1 ) for 24, 48, and 72 h. The cells were subjected to specific parameters of phototherapy (PT) (LASERTable-InGaAsP-780 ± 3 nm, 25 mW, 3 J cm-2 ). Cell proliferation (alamarBlue® ), viability (Trypan Blue) and synthesis of CCL2 (ELISA) were evaluated. Data were statistically analyzed by the Kruskal-Wallis and Mann-Whitney test (α = 5%). Proliferation and viability of both cell lines decreased after LPS treatment at 48 and 72 h. Enhanced synthesis of CCL2 by gingival fibroblasts occurred at 24 h, while epithelial cells increased synthesis of this chemokine at 48 and 72 h. PBM enhanced cell proliferation and viability in a time-dependent manner for both cell lines exposed or not to LPS, while synthesis of CCL2 by cells exposed to PT decreased over time. PBM caused biomodulatory effects on gingival fibroblasts and epithelial cells previously treated with LPS. These effects may decrease tissue inflammatory response and accelerate wound healing of oral mucosal tissue.

Epithelial cell-enhanced metabolism by low-level laser therapy and epidermal growth factor.

Reepithelialization and wound closure are the desired outcome for several ulcerative conditions. Such resolution reduces the possibility of wound contamination and maintenance of the injury and improves the reestablishment of tissue morphology and functions. Investigators are seeking adjuvant therapies that can accelerate wound healing and are developing new strategies for clinical applications. This study compared the effects of epidermal growth factor (EGF) application and low-level laser therapy (LLLT) on cultured epithelial cells. Cells were seeded in 24-well plates. After a 24-h incubation, the epithelial cells were either treated with EGF (100 µM in serum-free DMEM for 72 h) or subjected to LLLT (780 nm, 25 mW, 0.5, 1.5, and 3 J/cm2) by three applications every 24 h. Seventy-two hours after cells were treated with EGF or LLLT, cell migration, viability, proliferation, and collagen synthesis were assessed. Cells treated with EGF showed increased cell viability, proliferation, and collagen synthesis compared with those cells that received no treatment. LLLT enhanced cell migration; however, no significant effects of laser irradiation on other cell functions were observed. Comparison of both therapies demonstrated that EGF and LLLT enhanced specific epithelial cell activities related to wound healing.

Effects of low-level laser therapy and epidermal growth factor on the activities of gingival fibroblasts obtained from young or elderly individuals.

This study evaluated the effects of low-level laser therapy (LLLT) and epidermal growth factor (EGF) on fibroblasts obtained from young and elderly individuals. Gingival fibroblasts from young (Y) and elderly (E) individuals were seeded in wells of 24-well plates with Dulbecco's modified Eagle's medium (DMEM) containing 10 % of fetal bovine serum (FBS). After 24 h, the cells were irradiated (LASERTable-InGaAsP-780 ± 3 nm, 25 mW, 3 J/cm2) or exposed to EGF (100 µM). After 72 h, cells were evaluated for viability, migration, collagen and vascular endothelial growth factor (VEGF) synthesis, and gene expression of growth factors. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (α = 5 %). Y and E fibroblasts irradiated with laser or exposed to EGF showed increased viability and collagen synthesis. Enhanced cell migration was observed for Y fibroblasts after both treatments, whereas only the LLLT stimulated migration of E cells. VEGF synthesis was higher for Y and E cells exposed to EGF, while this synthesis was reduced when E fibroblasts were irradiated. Increased gene expression of VEGF was observed only for Y and E fibroblasts treated with LLLT. Regardless of a patient's age, the LLLT and EGF applications can biostimulate gingival fibroblast functions involved in tissue repair.

Response of a co-culture model of epithelial cells and gingival fibroblasts to zoledronic acid.

Osteonecrosis of the jaw is an adverse effect of bisphosphonates. While the etiopathogenesis of this condition has been investigated, the interactions and effects of bisphosphonates on oral mucosa cells remain unclear. It is hypothesized that cell culture models, such as co-culture or three-dimensional cell culture models, can provide valuable insight. Therefore, the aim of this study was to evaluate the effects of zoledronic acid (ZA) on epithelial cells and gingival fibroblasts in a co-culture model. Briefly, epithelial cells were seeded on transwell inserts and gingival fibroblasts were seeded in the lower well of 24-well plates. The latter were treated with ZA (5 µM) for 24 or 48 h. Cell viability and synthesis of the inflammatory chemokine, CCL2, were subsequently assessed. Data were subjected to statistical analysis with a 5% significance level. In the presence of ZA, the epithelial cells exhibited significant toxicity in both cell culture models and at both time points. However, greater cytotoxicity was observed in the co-culture model. Greater viability for the gingival fibroblasts was also associated with the co-culture model, and ZA-mediated toxicity was observed for the 48 h time point. ZA promoted a significant increase in CCL2 synthesis in both sets of cells, with greater CCL2 synthesis detected in the gingival fibroblasts. However, this effect was diminished in the co-culture model. Taken together, these results confirm the specific response patterns of the cells seeded in the co-culture model and also demonstrate the protective mechanism that is mediated by epithelial/mesenchymal cell interactions upon exposure to ZA.

Functional Differences In Gingival Fibroblasts Obtained from Young and Elderly Individuals.

Fibroblasts participate in the wound repair process through proliferation and migration as well as the synthesis of factors growth and extracellular matrix molecules. However, cell aging and the individual himself can lead to reduction of cell functions and consequently, the ability of tissue repair. This study evaluated the activity of gingival fibroblasts from young (Y) and elderly (Y) patients and their responsiveness to tumor necrosis factor alpha (TNF-a). Gingival fibroblasts were isolated from six patients (3Y; and 3E) and seeded in complete culture medium (DMEM). For cell viability analysis, total protein production and collagen synthesis, fibroblasts were cultured in 96-well plates for 24, 48 or 72 h (n=36). Cell responses to TNF-a, was evaluated by application of this cytokine to cultured cells (100 ng/mL) for 24 h, followed by evaluation of reactive oxygen species (ROS), nitric oxide (NO) and CCL5 production (n=36). Data were analyzed by Kruskal-Wallis and the Mann-Whitney U tests (a = 0.05). Viability of E fibroblasts was higher than Y fibroblasts for 24 and 48 h, but these cells showed gradual reduction of viability over the course of time. For Y cells, reduced collagen synthesis was observed at 48 h. No difference was observed in ROS production for both cells after TNF-a exposure. However, both cultures showed increased production of NO and CCL5 in the presence of TNF-a. Functional differences and distinct responsiveness to TNF-a were observed according to patient's age.