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%)

The Minimal Clinically Relevant Change of the FOG Score.

BACKGROUND: Freezing of gait is a highly disabling symptom in persons with Parkinson's disease (PwP). Despite its episodic character, freezing can be reliably evaluated using the FOG score. The description of the minimal clinically relevant change is a requirement for a meaningful interpretation of its results. OBJECTIVE: To determine the minimal clinically relevant change of the FOG score. METHODS: We evaluated video recordings of a standardized freezing-evoking gait parkour, i.e., the FOG score just before and 30 minutes after the intake of a regular levodopa dose in a randomized blinded fashion. The minimal clinically relevant response was considered a value of one or more on a 7-step Likert-type response scale [-3; +3] that served as the anchor. The minimal clinically relevant change was determined by ROC analysis. RESULTS: 37 PwP (Hoehn & Yahr stages 2.5-4, 27 male, 10 female) were aged 68.2 years on average (range 45-80). Mean disease duration was 12.9 years (2-29 years). Minimum FOG score was 0 and Maximum FOG score was 29. Mean FOG scores before medication were 10.6, and 11.1 after medication intake, with changes ranging from -14.7 to +16.7. The minimal clinically relevant change (MCRC) for improvement based on expert clinician rating was three scale points with a sensitivity of 0.67 and a specificity of 0.96. CONCLUSIONS: The FOG score is recognized as a useful clinical instrument for the evaluation of freezing in the clinical setting. Knowledge of the MCRC should help to define responses to interventions that are discernible and meaningful to the expert physician and to the patient.

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.

Biomechanical strain-induced modulation of proliferation coincides with an ERK1/2-independent nuclear YAP localization.

Biomechanical strain induces activation of the transcriptional co-activator yes-associated protein (YAP) by nuclear re-distribution. Recent findings indicate that the mechanically responsive mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) 1/2 is involved in the amount of nuclear YAP, reflecting its activation. In this context, we conducted experiments to detect how biomechanical strain acts on the subcellular localization of YAP in periodontal cells. To this end, cells were subjected to 2.5% static equiaxial strain for different time periods. Western blot and fluorescence imaging-based analyses revealed a clear modulation of nuclear YAP localization. This modulation fairly coincided with the altered course of the KI-67 protein amount in conjunction with the percentage of KI-67-positive and thus proliferating cells. The inhibition of the ERK1/2 activity via U0126 yielded an unchanged strain-related modulation of nuclear YAP localization, while YAP amount in whole cell extracts of strained cells was decreased. Administration of the YAP-inhibiting drug Verteporfin evoked a clear reduction of KI-67-positive and thus proliferating cells by approximately 65%, irrespective of strain. Our data reveal YAP as a regulator of strain-modulated proliferation which occurs in a MAPK-independent fashion.

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.

Modulation of focal adhesion constituents and their down-stream events by EGF: On the cross-talk of integrins and growth factor receptors.

Within the concept of integrin growth factor receptor (GFR) cross-talk, little is known about the effects of GFRs on focal adhesions (FAs). Therefore, we tested the hypothesis whether EGF can modulate constituents of FAs and subsequent down-stream events. To this end, EGF-treated keratinocytes were subjected to combined fluorescence imaging and western blotting, to quantify expression and/or activation of molecules, involved in integrin GFR cross-talk, and receptor proximal and distal signaling events. Generally, EGF response revealed an amplified redistribution or activation of molecules under study, which will be explained in detail from the plasma membrane to the cell interior. In addition to significant activation of EGF receptor (EGFR) at tyrosine Tyr845, a remarkable redistribution was detectable for the focal adhesion constituents, integrin ß1 and ß3, and zyxin. Increased activation also applied to focal adhesion kinase (FAK) by phosphorylation at Tyr397, Tyr576, and Src at Tyr418, while total FAK remained unchanged. Risen activity was seen as well for the analyzed distal down-stream events, p190RhoGAP and MAP kinases p42/44. Intriguingly, Src-specific inhibitor Herbimycin A abrogated the entire EGF response except FAK Tyr397 phosphorylation, independent of EGF presence. Mechanistically, our results show that EGF modulates adhesion in a dual fashion, by firstly redistributing focal adhesion constituents to adhesion sites, but also by amplifying levels of activated RhoA antagonist p190RhoGAP, important for cell motility. Further, the findings suggest that the observed EGF response underlies an EGFR integrin cross-talk under recruitment of receptor proximal FAK and Src, and MAP kinase and p190RhoGAP as receptor distal events.

Focal adhesion kinase (FAK) perspectives in mechanobiology: implications for cell behaviour.

Mechanobiology is a scientific interface discipline emerging from engineering and biology. With regard to tissue-regenerative cell-based strategies, mechanobiological concepts, including biomechanics as a target for cell and human mesenchymal stem cell behaviour, are on the march. Based on the periodontium as a paradigm, this mini-review discusses the key role of focal-adhesion kinase (FAK) in mechanobiology, since it is involved in mediating the transformation of environmental biomechanical signals into cell behavioural responses via mechanotransducing signalling cascades. These processes enable cells to adjust quickly to environmental cues, whereas adjustment itself relies on the specific intramolecular phosphorylation of FAK tyrosine residues and the multiple interactions of FAK with distinct partners. Furthermore, interaction-triggered mechanotransducing pathways govern the dynamics of focal adhesion sites and cell behaviour. Facets of behaviour not only include cell spreading and motility, but also proliferation, differentiation and apoptosis. In translational terms, identified and characterized biomechanical parameters can be incorporated into innovative concepts of cell- and tissue-tailored clinically applied biomaterials controlling cell behaviour as desired.

Nonwoven-based gelatin/polycaprolactone membrane proves suitability in a preclinical assessment for treatment of soft tissue defects.

Standard preclinical assessments in vitro often have limitations regarding their transferability to human beings, mainly evoked by their nonhuman and tissue-different/nontissue-specific source. Here, we aimed at employing tissue-authentic simple and complex interactive fibroblast-epithelial cell systems and their in vivo-relevant biomarkers for preclinical in vitro assessment of nonwoven-based gelatin/polycaprolactone membranes (NBMs) for treatment of soft tissue defects. NBMs were composed of electrospun gelatin and polycaprolactone nanofiber nonwovens. Scanning electron microscopy in conjunction with actin/focal contact integrin fluorescence revealed successful adhesion and proper morphogenesis of keratinocytes and fibroblasts, along with cells' derived extracellular matrix deposits. The "feel-good factor" of cells under study on the NBM was substantiated by forming a confluent connective tissue entity, which was concomitant with a stratified epithelial equivalent. Immunohistochemistry proved tissue authenticity over time by abundance of the biomarker vimentin in the connective tissue entity, and chronological increase of keratins KRT1/10 and involucrin expression in epithelial equivalents. Suitability of the novel NBM as wound dressing was evidenced by an almost completion of epithelial wound closure in a pilot mini-pig study, after a surgical intervention-caused gingival dehiscence. In summary, preclinical assessment by tissue-authentic cell systems and the animal pilot study revealed the NBM as an encouraging therapeutic medical device for prospective clinical applications.

Natural corneal cell-based microenvironment as prerequisite for balanced 3D corneal epithelial morphogenesis: a promising animal experiment-abandoning tool in ophthalmology.

To achieve durable recognition as a promising animal experiment-abandoning tool in ophthalmology, in vitro engineered tissue equivalents of the human cornea should exhibit proper morphogenesis. Regarding this issue, we were seeking for the natural cell microenvironment fulfilling the minimum requirements to allow human corneal keratinocytes to develop a balanced epithelial morphology with regular spatial appearance of tissue homeostatic biomarkers. Hence, we established cocultures of 3D cell-based collagen scaffolds comprising immortalized corneal keratinocytes combined with a gradual cornea-derived in vivo-like cell microenvironment, together with immortalized stromal fibroblasts alone (nonholistic) or fibroblasts and immortalized endothelial cells (holistic). With matched non-holistic microenvironments revealing mostly flattened cells and putative apical cell ablation foci at day 6, and 9 in HE stains, holistic counterparts yielded proper epithelial stratification with cell flattening restricted to apical layers. Concordantly, RT(2)-PCR showed a tremendous increase in gene expression for progressive and terminal biomarkers of corneal keratinocyte differentiation, cytokeratin (CK) 12, and filaggrin (FIL), in response to nonholistic environments, while involucrin (INV) was moderately but significantly upregulated. Although visible, this increase was moderate in corneal keratinocytes with a holistic environment. On the protein level, indirect immunofluorescence revealed that only epithelia of holistic environments showed diminishment in CK19, counteracted by CK12 rising over time. This time-dependent progression in differentiation coincided with declined proliferation and tissue-regular focus of differentiation biomarkers inv and fil to suprabasal and apical cell layers. Our novel findings suggest the interplay of native tissue forming cell entities, important for balanced corneal epithelial morphogenesis. In addition, they provide evidence for a holistic cell microenvironment as a prerequisite for development of an in vitro engineered corneal epithelial tissue equivalent, exhibiting a regular appearance of tissue homeostatic biomarkers. Such equivalents will be promising tools in ophthalmology, for example, for mechanistic studies in basic research and/or testing of generics or preclinical validation of innovative cornea-tailored biomaterials, desired for regenerative strategies.

Generation and evaluation of a human corneal model cell system for ophthalmologic issues using the HPV16 E6/E7 oncogenes as uniform immortalization platform.

The present study aimed at employing the human papillomavirus type 16 (HPV16) E6/E7 gene platform, to create a uniform authentic in vitro model cell system of the human cornea for ophthalmologic issues and here especially for prospective biomaterial evaluations for therapeutic regenerative approaches. Therefore, HPV16 E6/E7 genes were employed as uniform platform to immortalize primary human corneal keratinocytes (IHCK), fibroblasts (IHCF), and endothelial (IHCE) cells. qPCR revealed that E6/E7 mRNA transcription persisted at rising passages and FISH detection of the chromosome portfolio 1, 8, 10 and 18 showed fairly the disomic cytogenetic status. Hot spot passages proved oscillation of aneuploidies in the entire passage spectrum under study, while hot spot aneuploidies annotated prevalence for distinct chromosomes. Though IIF revealed general endurance, tissue-innate corneal biomarkers were modulated, i.e. expressed in a temporal-confluence, temporal-spatial or passage-dependent manner. In summary, by the fairly normal chromosomal status, and expression of tissue-innate biomarkers, we created for the first time a uniform authentic in vitro model cell system of the human cornea, by application of the HPV16 E6/E7 immortalization platform only. This system renders a precious tool for prospective iterative in vitro studies on issues such as corneal tissue homeostasis, pharmaceutical generics, and/or evaluation of new biomaterials for clinical corneal applications.

Multi-chromatic control of mammalian gene expression and signaling.

The emergence and future of mammalian synthetic biology depends on technologies for orchestrating and custom tailoring complementary gene expression and signaling processes in a predictable manner. Here, we demonstrate for the first time multi-chromatic expression control in mammalian cells by differentially inducing up to three genes in a single cell culture in response to light of different wavelengths. To this end, we developed an ultraviolet B (UVB)-inducible expression system by designing a UVB-responsive split transcription factor based on the Arabidopsis thaliana UVB receptor UVR8 and the WD40 domain of COP1. The system allowed high (up to 800-fold) UVB-induced gene expression in human, monkey, hamster and mouse cells. Based on a quantitative model, we determined critical system parameters. By combining this UVB-responsive system with blue and red light-inducible gene control technology, we demonstrate multi-chromatic multi-gene control by differentially expressing three genes in a single cell culture in mammalian cells, and we apply this system for the multi-chromatic control of angiogenic signaling processes. This portfolio of optogenetic tools enables the design and implementation of synthetic biological networks showing unmatched spatiotemporal precision for future research and biomedical applications.

A red/far-red light-responsive bi-stable toggle switch to control gene expression in mammalian cells.

Growth and differentiation of multicellular systems is orchestrated by spatially restricted gene expression programs in specialized subpopulations. The targeted manipulation of such processes by synthetic tools with high-spatiotemporal resolution could, therefore, enable a deepened understanding of developmental processes and open new opportunities in tissue engineering. Here, we describe the first red/far-red light-triggered gene switch for mammalian cells for achieving gene expression control in time and space. We show that the system can reversibly be toggled between stable on- and off-states using short light pulses at 660 or 740 nm. Red light-induced gene expression was shown to correlate with the applied photon number and was compatible with different mammalian cell lines, including human primary cells. The light-induced expression kinetics were quantitatively analyzed by a mathematical model. We apply the system for the spatially controlled engineering of angiogenesis in chicken embryos. The system's performance combined with cell- and tissue-compatible regulating red light will enable unprecedented spatiotemporally controlled molecular interventions in mammalian cells, tissues and organisms.

Interactive fibroblast-keratinocyte co-cultures: an in vivo-like test platform for dental implant-based soft tissue integration.

For clinicians, soft connective tissue integration (STI), one of the critical issues for dental implant success, is usually tested using the fibroblasts monolayer regime. Therefore, we aimed at an extension of this regime by employing interactive gingival fibroblast-keratinocyte cocultures (CCs) as an in vivo-like test platform. In the extended regime, 13 STI-relevant genes were analyzed in response to five different titanium implant biomaterial surfaces. The genes quantitated by real-time polymerase chain reaction were categorized as pro supportive or contra supportive, that is, nonsupportive for cell growth on an engineered surface. Monocultures had higher levels of contra supportive gene expression, but the fibroblast-keratinocyte CC had two out of five of the titanium surfaces with more pro supportive gene expression than contra supportive gene expression. We defined this change from contra supportive gene expression to pro supportive gene expression by developing the "relative supportive difference" index. Hence, interactive CCs exhibit valuable supportive effects on the expression of STI-relevant genes, possibly via physiological cell-to-cell-interactions. Our results render interactive gingival CCs suitable as a test platform for dental implant-related STI under more in vivo-like conditions.

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.