Cytotoxic and inflammatory response of human lung epithelial cells A549 to particles released from dental restorative materials during dry and wet grinding.

OBJECTIVES: The aim was to evaluate the release of particles from dental materials during wet and dry grinding and test their effects on human lung epithelia cells in-vitro. METHODS: Four dental restorative materials were used: two composites [Ceram.x® universal (Dentsply Sirona) and Filtek™ Supreme XTE (3 M)], one ceramic [VITABLOCS® Mark II (VITAy)] and a ceramic-resin material [Lava™ Ultimate (3 M)]. Material samples were ground to powder under standardized wet and dry conditions in an isolated dental room. During grinding, the particle concentrations were measured with LAS and CPC. Baseline values were measured before grinding. The particles' size was evaluated using DLS and SEM. Water was used as control. The cytotoxicity and inflammatory response of the lung cells (A549) after exposure to different concentrations (1, 3, 10, 30, 100, 300 µg/mL) of the generated dust were analyzed with LDH, WST-1 and ELISA. RESULTS: LAS and CPC revealed a high concentration of particles< 10 µm and< 1 µm respectively, into the air. Particles showed high tendency to agglomerate. DLS showed particle size distribution between 150 nm and 18 µm independently of the material composition. All materials induced significant effects (p < 0.05) on the cell membrane integrity and viability of the A549 cells. Only the ceramic particles showed a significant increase in hydroxyl radical formation at low concentrations (p < 0.05), for both wet and dry conditions. All materials except ceramic, induced a significant release of IL-8 in A549 cells at 300 µg / mL (p < 0.05). SIGNIFICANCE: Wet and dry grinding of dental materials result in release of ultrafine and fine particulate matter into the air. The in-vitro findings on the cellular response of lung cells to generated dust indicate a potential risk for human health due inhalation of the released particles. The use of water-cooling seems to be beneficial resulting in reduced release of particles compared to dry grinding.

Response of human gingival keratinocytes to hybrid CAD/CAM material eluates.

OBJECTIVES: The aim of this study was to investigate the influence of hybrid CAD/CAM-blocks on immortalized human gingival keratinocytes (HGK). METHODS: Samples of two different hybrid CAD/CAM materials [Lava™ Ultimate (3 M); VITA Enamic® (VITA Zahnfabrik)], a composite material [ceram.x® universal (Dentsply Sirona)] and a CAD/CAM ceramic [VITABLOCS® (VITA Zahnfabrik)] were stored in cell culture medium for 72 h to prepare eluates according to ISO-10993-12:2012. HGK were exposed to eluates for 6, 24 and 48 h. Cell monitoring was performed by RTCA iCELLigence™ system. The morphological changes were evaluated using phase contrast imaging. Specific biomarkers of apoptosis and terminal differentiation (Caspase-3, Involucrin) were analyzed semi quantitatively by indirect immunofluorescence (IIF). Protein levels and activation of MAP kinases ERK1/2 (p44/42) were quantified by Western blot. Data were statistically analyzed by unpaired t-test (p < 0.05). RESULTS: Regarding Vita Enamic® and Lava™ Ultimate, results of RTCA iCELLigence™ and Western blots showed no statistically significant differences (p > 0.05) compared to the negative control (HGK in native keratinocyte growth medium). No aberrant expression of Caspase-3 and Involucrin was detected in cells incubated with Vita® Enamic eluates Cells incubated with Lava™ Ultimate showed a higher expression of Involucrin after 24 h of incubation compared to the negative control. Statistically significant differences (p < 0.01) were found between cells incubated with ceram.x® universal and the negative control in RTCA iCELLigence™ assay and in quantitative measurements of Western blots after 6 h against phospho-p44/42 (p = 0.044). Increased expression of Caspase-3 and Involucrin were detected by IIF in cells after incubation with eluates of ceram.x® universal. SIGNIFICANCE: The present data show no significant effect of hybrid materials on analyzed functions of cell behavior. A cytotoxic influence of ceram.x® universal eluates was observed in HGK in terms of a strong modulation of proliferation, morphology and protein expression.

Novel In Situ-Cross-Linked Electrospun Gelatin/Hydroxyapatite Nonwoven Scaffolds Prove Suitable for Periodontal Tissue Engineering.

Periodontal diseases affect millions of people worldwide and can result in tooth loss. Regenerative treatment options for clinical use are thus needed. We aimed at developing new nonwoven-based scaffolds for periodontal tissue engineering. Nonwovens of 16% gelatin/5% hydroxyapatite were produced by electrospinning and in situ glyoxal cross-linking. In a subset of scaffolds, additional porosity was incorporated via extractable polyethylene glycol fibers. Cell colonization and penetration by human mesenchymal stem cells (hMSCs), periodontal ligament fibroblasts (PDLFs), or cocultures of both were visualized by scanning electron microscopy and 4',6-diamidin-2-phenylindole (DAPI) staining. Metabolic activity was assessed via Alamar Blue® staining. Cell type and differentiation were analyzed by immunocytochemical staining of Oct4, osteopontin, and periostin. The electrospun nonwovens were efficiently populated by both hMSCs and PDLFs, while scaffolds with additional porosity harbored significantly more cells. The metabolic activity was higher for cocultures of hMSCs and PDLFs, or for PDLF-seeded scaffolds. Periostin and osteopontin expression was more pronounced in cocultures of hMSCs and PDLFs, whereas Oct4 staining was limited to hMSCs. These novel in situ-cross-linked electrospun nonwoven scaffolds allow for efficient adhesion and survival of hMSCs and PDLFs. Coordinated expression of differentiation markers was observed, which rendered this platform an interesting candidate for periodontal tissue engineering.

Low-dose Bisphenol A and its analogues Bisphenol F and S activate estrogen receptor ß and slightly modulate genes in human gingival keratinocytes.

OBJECTIVES: This study investigated the putative activation of estrogen receptor ß (ERß) and possible effects related on gene expression in oral mucosal cells in response to the endocrine disruptor Bisphenol A (BPA) and its analogues Bisphenol F (BPF) and Bisphenol S (BPS). METHODS: Human gingival keratinocytes (HGK) were exposed to BPA-, BPF-, and BPS-solutions in concentrations of 1.3 µM, 0.16 µM and 11.4 nM as well as 200 pM and 100 nM estradiol (E2) for 6 h, 24 h and 4 d. Indirect immunofluorescence (IIF) was performed to detect a possible ERß activation. Additionally, transcription of keratinocyte-relevant biomarkers was analyzed by quantitative real-time PCR (qRT-PCR). A linear mixed model and pairwise comparisons were applied for statistical analyses. RESULTS: The tested concentrations of BPA, BPF, BPS and E2 revealed distinct activation of ERß at all time periods, whereat 100 nM E2 induced the most pronounced activation. Despite the detected ERß activation, the concentrations of BPA and its analogues induced only moderate modulation of the tested keratinocyte-relevant biomarker genes at all time periods. This also applied to 200 pM E2, while in case of 100 nM E2 significant changes (p < 0.05) were detected for almost all analyzed genes. SIGNIFICANCE: Though BPA and its analogues induce activation of ERß irrespective from the chosen concentrations and incubation periods, they lack significant modulation of gene expression of keratinocyte-relevant biomarkers. Although limited to a selected number of genes, the sparse modulation of gene expression may give a hint that the substances do slightly affect transcription of gingival-keratinocyte-innate genes, since the concentrations applied to HGK were of physiological importance.

Detection of Bisphenol A in dental wastewater after grinding of dental resin composites.

OBJECTIVES: This study evaluated the release of bisphenol A (BPA) in wastewater after grinding of resin composites and tested three filtration materials. METHODS: Three resin composites (Ceram X, Filtek Supreme XTE and Core-X flow) were used. Samples (5mm×2mm, n=10) were prepared using a metal mold and were polymerized for 20s according to manufacturers' instructions. A dental unit was disconnected from wastewater circulation and composite samples were ground under standardized procedures (200,000rpm; 90s). Wastewater was collected in glass bottles. Water samples were collected as control by performing the same procedure without grinding resin composite. All samples were stored at 7°C for 6 months to simulate storage. Then they were analyzed by HPLC-FLD. Three filtration materials (Zeosorb, Katalox Light and Catalytic Carbon) were used for water treatment to remove BPA. BPA-water solutions were prepared; corresponding to the highest amount released by the resin composites. These solutions were analyzed before and after filtration by HPLC-FLD and their efficacy (%) was calculated. RESULTS: BPA was detected in all composite solutions: Ceram X and Filtek Supreme XTE showed similar findings (p>0.05) which were significantly higher than the control (p<0.001) and Core-X flow (p=0.001). The efficacy of the filtration materials was: Katalox Light (5.09%)

Nanofibered Gelatin-Based Nonwoven Elasticity Promotes Epithelial Histogenesis.

Regarding tissue regeneration, mechanics of biomaterials gains progressive importance. Therefore, this study reports on in situ crosslinked electrospun gelatin nonwoven mats (NWMs) whose distinct modulus of elasticity (ME) promotes epithelial tissue formation in a graded manner. NWMs, comprising fiber diameters in various distributions, yield an ME of about 2.1, 3.2, and 10.9 kPa. A two-step approach of preclinical in vitro validation identifies the elasticity of 3.2 kPa as superior to the other, regarding the histogenetic epithelial outcome. Hence, this 3.2 kPa candidate NWM is colonized with oral mucosal epithelial keratinocytes in the absence or presence of mesenchymal fibroblasts and/or endothelial cells. Evaluation of epithelial histogenesis at days 1 to 10 occurs by colorimetric and fluorescence-based immunohistochemistry (IHCH) of specific biomarkers. These include cytokeratins (CK) 14, CK1, and involucrin that indicate different stages of epithelial differentiation, as well as the basement membrane constituent collagen type IV and Ki-67 as a proliferation marker. Intriguingly, histogenesis and IHCH reveal the best resemblance of the native epithelium by the NWM alone, irrespective of other cell counterparts. These findings prove the gelatin NWM a convenient cell matrix, and evidence that NWM mechanics is important to promote epithelial histogenesis in view of prospective clinical applications.

Disruption of adherens junction and alterations in YAP-related proliferation behavior as part of the underlying cell transformation process of alcohol-induced oral carcinogenesis.

Accumulating evidences indicate that alcohol might play a causative in oral cancer. Unfortunately, in vitro cell systems, uncovering the molecular background of the underlying cell transformation process, are rare. Therefore, this study was conducted, to identify molecular changes and characterize their putative cell behavioral consequences in epitheloid (EPI) and fibroblastoid (FIB) oral keratinocyte phenotypes, arising from chronical alcohol treatment. Concerning adherens junctions (AJs), both EPI and FIB showed membrane-bound ß-catenin, but exhibited differences for E-cadherin and zyxin. While EPI revealed E-cadherin/ß-catenin membrane co-localization, which in parts also applied for zyxin, FIB membranes were devoid of E-cadherin and exhibited marginal zyxin expression. Fetal calf serum (FCS) administration in starved cells promoted proliferation in both keratinocyte phenotypes, whereat EPI and FIB yielded a strikingly modified FCS sensitivity on the temporal scale. Impedance measurement-based cell index detection yielded proliferation stimulation occurring much earlier in FIB (<20h) compared to EPI (>45h). Nuclear preference of the proliferation-associated YAP co-transcription factor in FIB was FCS independent, while it required FCS in EPI. Taken together, the lack of membrane-inherent E-cadherin/ß-catenin co-localization together with low zyxin - reveals perturbation of AJ integrity in FIB. Regarding cell behavior, perturbed AJs in FIB correlate with temporal proliferation sensitivity towards FCS. CYF of 5.6 strongly suggests involvement of chromatin-bound YAP in FIB's proliferation temperosensitivity. These molecular differences detected for EPI and FIB are part of the underlying cell transformation process of alcohol-induced oral carcinogenesis, and indicate FIB being in a more advanced transformation stage.

Effects of low-dose Bisphenol A on calcium ion influx and on genes of proliferation and differentiation in immortalized human gingival cells in vitro: The role of estrogen receptor beta.

OBJECTIVES: Relating to low-dose Bisphenol-A (BPA), there is still a lack of mechanistic studies in oral cells, representing the first targets of BPA by oral intake. The objective of this study was to investigate an assumed mechanistic interrelationship between both low-dose BPA-modulated Calcium ion (Ca2+) influx and cell behavior, and the estrogen receptor ß (ERß), in oral mucosal cells. METHODS: Indirect immunofluorescence (IIF) was conducted on estrogen receptor beta (ERß) activity after 1, 3, and 6days in response to 39nM BPA, 15µM BPA, and 200 pM 17ß-Estradiol (E2). In addition to Ca2+ concentration measurement, qPCR for proliferation and differentiation biomarkers was performed, to examine cell behavior. Fulvestrant-mediated ER inhibition was employed to seek for a mechanistic role of ERß in regulating BPA-emanating effects. RESULTS: While both E2 and BPA yielded ERß activation, 39nM BPA and 200 pM E2 did not change MKI67 proliferation marker expression, but reduced transcription of differentiation markers. Conversely, 15µM BPA reduced MKI67 transcription, but significantly increased differentiation gene expression and intracellular Ca2+ levels. Fulvestrant-induced ERß inhibition yielded complete elimination of all E2- and BPA-triggered modulatory effects, suggesting a mechanistic role of activated ERß for BPA-mediated Ca2+ influx and keratinocyte differentiation. SIGNIFICANCE: Concerning cell behavior, these findings provide significant evidence of a threshold-dependent transcription of proliferation and differentiation-related genes as well as Ca2+ influx in response to 39nM and 15µM low-dose BPA, which identify a mechanistic role of activated ERß in oral keratinocytes.

Gene expression analysis of conventional and interactive human gingival cell systems exposed to dental composites.

OBJECTIVES: The aim of this study was the detection of putative gene expression-related effects of dental composites in conventional and interactive gingival cell systems. METHODS: Conventional monoculture (MC) and interactive cell systems (ICS) comprising human gingival fibroblast (HGF) and immortalized human gingival keratinocytes (IHGK) were exposed for 24h and 7 days according to ISO10993-12:2012 manufactured eluates of different composites (Ceram X(®), Filtek™ Supreme XT, Filtek™ Silorane, Fusio™ Liquid Dentin, and Vertise™ Flow). qRT-PCR-based mRNA analysis for biomarkers indicating cell proliferation, differentiation, apoptosis, inflammation, and adhesion was performed. Apoptotic cells were quantified by annexin-V labeling. RESULTS: Due to low RNA amounts, qPCR could not be performed for Vertise™ Flow and Fusio™ Liquid Dentin at day 7. At 24h, flowables yielded increased transcription for biomarkers of inflammation and apoptosis in IHGK, irrespective of the cell system. HGF cultures displayed lower transcription for cell adhesion markers in both cell systems. Filtek™ Supreme XT showed increased differentiation by elevated filaggrin gene expression in both cell systems for IHGK at day 7, while Filtek™ Silorane and Ceram X(®) yielded elevation of inflammation biomarkers in both cell types. Annexin-V labeling revealed high apoptosis rates for both flowables and Filtek™ Supreme XT for IHGK, while low rates were detected for Filtek™ Silorane and Ceram X(®). SIGNIFICANCE: Among the composites evaluated, exposition of IHGK and HGF in conventional and interactive cell systems demonstrated most pronounced gene expression alterations in response to flowables, coinciding with elevated levels of apoptosis.

Human gingival keratinocyte response to substances eluted from silorane composite material reveal impact on cell behavior reflected by RNA levels and induction of apoptosis.

OBJECTIVES: The aim of this study was the characterization of siloran-derived composite eluates in conjunction with their putative impact on human gingival keratinocytes (HGK), i.e. levels of total RNA and induction of apoptosis compared to a methacrylate-based material. METHODS: Standardized Filtek™ Silorane specimens (n = 20) were subjected to scanning ion monitoring to detect monomer masses between 100 and 1000, after storage in human saliva, and 75% ethanol for up to 28 days. In order to evaluate the effect on cells, HGK were exposed to eluates from Filtek™ Silorane, Filtek™ Supreme XT and control medium for 1 and 4 days, prior to isolation of total RNA, and Annexin-5 fluorescence labeling indicating induction of apoptosis. RESULTS: Irrespective of the mode and storage time, SIM identified discrete peaks, corresponding to masses of "393" and "337". In response to both composite eluates, an effect on HGK was reflected by drastically reduced levels of isolated total RNA at each time period (after 1 day: control: 302 ng/µl; Filtek™ Silorane: 128 ng/µl, Filtek™ Supreme XT: 129 ng/µl and after 4 days: control: 528 ng/µl; Filtek™ Silorane: 162 ng/µl, Filtek™ Supreme XT: 166 ng/µl). Exposure to eluates from both composite materials yielded apoptosis induction in HGK, as demonstrated by a significant increase of cells exhibiting Annnexin-5 fluorescence. SIGNIFICANCE: Two distinct peaks were identified, which indicated the presence of corresponding substances. The composite-derived effects on HGK strongly suggest a negative impact on cells, as revealed by a clear reduction of total RNA levels, and significant increase in induction of apoptosis.