Minor salivary gland stem cells: a comparative study of the biological properties under clinical-grade culture conditions.

Development of clinical-grade, cell preparations is central to cGMP (good manufacturing practice compliant) conditions. This study aimed to investigate the potential of two serum/xeno-free, cGMP (StemPro, StemMacs) culture media to maintain "stemness" of human minor salivary gland stem cell (mSG-SC) cultures compared to a complete culture medium (CCM). Overall, StemMacs resulted in higher proliferation rates after p.6 compared to the conventional serum-based medium, while StemPro showed substantial delays in cell proliferation after p.9. The mSG-SCs cultures exhibited two distinct cell populations at early passages a mesenchymal subpopulation and an epithelial-like subpopulation. Expression of several markers (CD146, STRO-1, SSEA-4, CD105, CD106, CD34, K 7/8, K14, K18) variably decreased with prolonged passaging (all three media). The percentage of SA-ß-gal positive cells was initially higher for StemMacs compared to StemPro/CCM and increased with prolonged passaging in all cases. The telomere fragment length decreased with prolonged passaging in all three media but more pronouncedly for the CCM. Expansion under serum-free conditions caused pronounced upregulation of ALP and BMP-2, with parallel complete elimination of the baseline expressions of LPL (all three media) and ACAN (serum-free media), therefore, showing a preferential shift of the mSG-SCs towards osteogenic phenotypes. Finally, several markers (Nanog, SOX-2, PDX-1, OTX2, GSC, HCG) decreased with prolonged culture, indicating successive loss of "stemness". Based on the findings, it seems that StemPro preserve stemness of the mSG-SCs after prolonged culture. Nevertheless, there is still a vacant role for the ideal development of clinical-grade culture conditions.

Root coverage using a connective tissue graft with epithelial striation in combination with enamel matrix derivatives - a long-term retrospective clinical interventional study.

BACKGROUND: The application of a connective tissue graft with epithelial striation (CTG-ES) has been shown to improve the outcome of root coverage (RC) using the coronally advanced flap (CAF) and adjunctive administration of enamel matrix derivatives (EMD). Aim of the present study was to evaluate the long-term (mean: 16.19 ± 1.80 years, range: 13 to 18 years) stability of this treatment method with special focus on the location of the gingival margin and the width of keratinized tissue (WKT). METHODS: 16 patients (10 female, 6 male, aged 35.36 ± 14.70 years at surgery) with 25 Miller class I or II gingival recession (GR) defects were treated using the CAF combined with the CTG-ES and EMD. The clinical measurements recorded at baseline (t0), 6 months (t1), and 13 to 18 years (t2) after surgery included recession depth (RED), probing pocket depth (PPD), clinical attachment level (CAL), and WKT. In addition, the number of sites with complete RC (CRC) and the mean RC (MRC) were documented at t1 and t2. The statistical analysis was performed using a linear mixed model. RESULTS: The RED (t0: 4.52 ± 1.56 mm; t1: 0.36 ± 0.76 mm; t2: 0.30 ± 0.60 mm) and CAL (t0: 6.16 ± 1.62 mm; t1: 1.86 ± 0.87 mm; t2: 1.54 ± 0.92 mm) were significantly reduced at t1 and t2 compared to t0 (p <  0.001). The PPD was significantly reduced at t2 compared to t0 (p = 0.016). The WKT (t0: 1.18 ± 1.28 mm; t1: 3.26 ± 0.98 mm; t2: 4.26 ± 1.83 mm) significantly increased from t0 to t1, from t0 to t2 (p <  0.001) and from t1 to t2 (p = 0.007). A CRC was recorded at 19 sites (76.0%) at t1 and t2. The MRC was 93.6 ± 12.8% at t1 and 93.3 ± 13.3% at t2. CONCLUSIONS: The use of the CAF combined with CTG-ES and EMD leads to stable long-term outcomes on teeth with Miller Class I or II GR defects. The CTG-ES represents a hybrid graft with increased position stability and advantageous properties for the healing process. We assume that the ES is responsible for the increase of the WKT.

An early oral health care program starting during pregnancy--a long-term study--phase V.

OBJECTIVES: Objective was to analyze the effects of a long-term prevention program on dental and oral health of adolescents. MATERIALS AND METHODS: The entire study was subdivided into five phases. Phase I comprised an individual preventive care during pregnancy, phase II assessed mothers and their children until the age of 3, and in phase III until the age of 6. In phase IV, 13- to 14-year-old teenagers were investigated. In phase V, 18-19-year-old adolescents were examined (18.4 ± 0.4 years, n = 26). All phases consisted of an examination, education, and treatment based on the concept of an "early oral health care promotion." The control group consisted of randomly selected adolescents of the same age (n = 35). The following clinical parameters were assessed: DMF-T/DMF-S, HI, PBI, PSI, and Streptococcus mutans/lactobacilli concentration in saliva. RESULTS: The adolescents of the prevention group revealed a share of 92.3 % caries-free dentition. Mean DMF-T was 1.4 ± 2.6. The control group showed a significantly higher mean DMF-T of 3.8 ± 3.2 (p < 0.05) and revealed 71.4 % of caries-free dentition. The prevention group showed a significant lower PSI of 1.2 ± 0.8 compared to the control group (2.1 ± 0.4) (p < 0.05). CONCLUSION: An "early oral health care promotion" starting during pregnancy may cause a sustained and long-term improvement of the oral health of young adults. CLINICAL RELEVANCE: Prevention programs starting during pregnancy may establish an improved health behavior. Caries, periodontitis, and dietary complications in mother and child can be avoided by improving maternal oral health and by a tooth-friendly diet.

Oxidative stress is responsible for genotoxicity of camphorquinone in primary human gingival fibroblasts.

OBJECTIVES: The photoinitiator camphorquinone (CQ), used in dental restorative materials, was found to be cytotoxic in cell cultures. Previously, we have shown that CQ induces alkali labile sites and DNA strand breaks in human gingival fibroblasts (HGF) associated with an increase of intracellular reactive oxygen species (ROS). Therefore, the objective of our study was to evaluate if DNA damage in HGF cells is caused by the generation of ROS. MATERIAL AND METHODS: HGF cells were treated with different concentrations (0.5-2.5 mM) of CQ. The cell viability was assessed using propidium iodide (PI) assay. Oxidative DNA damage was evaluated by an enzyme-modified comet assay using human 8-hydroxyguanine DNA-glycosylase 1 (hOGG1), which converts oxidized 7,8-dihydro-8-oxoguanine (8-oxoguanine) into DNA strand breaks and functions as a marker for oxidative modified DNA. RESULTS: The results showed that CQ induced DNA damage in HGF cells without cytotoxic effects for the chosen treatment time. CQ treatment led to the generation of 8-oxoguanine in DNA, which can be shown by a significant increase in tail moment after CQ treatment by the enzyme-modified comet assay. CONCLUSION: It may be concluded that DNA damage due to CQ is caused by oxidative stress in gingival fibroblasts. CLINICAL RELEVANCE: A more detailed insight into genotoxic mechanisms in oral cells can be of great importance for a better understanding of the biocompatibility of CQ.

Does inhibition of proteolytic activity improve adhesive luting?

Endogenous enzymes may be involved in the biodegradation of adhesive restoration-tooth interfaces. Inhibitors of matrix metalloproteinases (MMPs) have been suggested to retard the bond-degradation process. Limited data are available on whether composite cements may also benefit from MMP inhibitors. Therefore, the aim of this study was to determine the effect of two MMP inhibitors--chlorhexidine digluconate (CHX) and galardin--on the microtensile bond strength (µTBS) of two self-adhesive composite cements to dentin. Ceramic specimens were cemented to bur-cut dentin surfaces using the self-adhesive composite cements RelyX Unicem 2 (3M ESPE) or Clearfil SA (Kuraray), or the etch-and-rinse composite cement Nexus 3 (Kerr) that served as the control. The surfaces were left untreated or were pretreated with MMP inhibitors (2% CHX or 0.2 mM galardin). The µTBS was determined 'immediately' and upon ageing (water storage for 6 months). Statistical analysis revealed a significant effect of the factors 'composite cement' and 'storage', as well as all interactions, but no effect of the MMP inhibitors. After 6 months of ageing, the µTBS decreased for all cements, except for the multistep etch-and-rinse luting composite when it was applied without MMP inhibitors. The MMP inhibitors could not prevent the decrease in µTBS upon ageing and therefore do not improve the luting durability of the composite cements tested.

Periodontal conditions in patients with Marfan syndrome - a multicenter case control study.

BACKGROUND: Marfan syndrome (MFS) is a disorder of the connective tissues. Alterations of the elastic fibers may manifest in different tissues especially in the skeletal, cardiovascular and ocular system. Oral manifestations like orthodontic or skeletal anomalies and fragility of the temporomandibular joint have been well described by various authors. However, no data are available regarding a possible periodontal involvement of MFS. Hence, the aim of the present study was to investigate for the first time if MFS may increase the susceptibility to periodontitis. METHODS: A comprehensive periodontal examination including documentation of probing pocket depth, gingival recession, clinical attachment level, and bleeding on probing was conducted in all patients. In addition, dental conditions were assessed by determining the Index for Decayed, Missing and Filled Teeth (DMFT) and a self-administered questionnaire was filled out by patients. For statistical analysis, the unpaired t-Test was applied (level of significance: p < 0.05). Both groups were matched concerning well known periodontal risk factors like age, gender and smoking habits. RESULTS: 82 participants, 51 patients with MFS (30 female and 21 male, mean age: 40.20 ± 15.35 years) and 31 sound controls (17 female and 14 male, mean age: 40.29 ± 13.94 years), were examined. All assessed periodontal and dental parameters were not significantly different between groups. CONCLUSIONS: Based on our data, patients with MFS did not reveal a higher prevalence of periodontitis compared to the control group. However, Marfan patients showed a tendency to more inflammation signs, which can be explained by the crowded teeth. Therefore, a regular professional cleaning of the teeth is recommendable (i.e., 6 months intervals) in order to reduce the bacterial biofilm in the oral cavity and thus resulting in a decreased risk of systemic diseases, specifically endocarditis.

Comparison of the push-out strength of two fiber post systems dependent on different types of resin cements.

The purpose of this study was to compare the push-out strength of glass fiber posts dependent on the resin cement. One hundred human teeth were divided into five groups (n = 20). Two glass fiber post systems (DT Light SL (DTSL) and RelyX Fiber Post (RF)) were used. DTSL posts were cemented with one "etch & rinse" system (ER) or one of three self-adhesive resin cements (SA). The RF posts were cemented with RelyX Unicem. Afterwards, half of the specimens were thermocycled (TC; 5°C/55°C, 5,000 cycles). All specimens were cut into disks (thickness, 2 mm). The push-out test was performed (crosshead speed, 1 mm/min), fracture types were determined (×25 and ×40 magnification), and statistical analysis was performed (one-way analysis of variance (ANOVA), Scheffe test, p < 0.05). One-way ANOVA showed a significant influence of the resin cement on the push-out strength of the glass fiber posts before thermocycling (p < 0.001). After TC, no significant differences were detected. Microscopic evaluation showed mainly adhesive failures between post and cement for ER or mixed fractures for SA. The bond strength of adhesively cemented glass fiber posts is not dependent on the type of resin cement after TC. The use of SA can lead to bond strength values comparable to ER. Self-adhesive resin cements could be used just as well as resin cements with "etch & rinse" adhesive systems for the cementation of glass fiber posts.

Assessment of the impact of two different isolation methods on the osteo/odontogenic differentiation potential of human dental stem cells derived from deciduous teeth.

Human deciduous teeth have been proposed as a promising source of mesenchymal stem cells for application in bone and dental tissue engineering. We established cultures of mesenchymal stem cells from the pulp of human deciduous teeth (deciduous teeth stem cells, DTSCs) and analyzed their morphologic, growth, immunophenotypic, and osteo/odontogenic differentiation characteristics using different isolation methods and culturing environments. We compared the biologic behavior of DTSCs isolated either by enzymatic dissociation (DTSCs-ED) or by direct outgrowth from pulp tissue explants (DTSCs-OG). We found that different isolation methods give rise to different populations/lineages of cells with respect to their phenotypic and differentiation characteristics. DTSCs-ED cultures comprised heterogeneous cell populations, whereas DTSCs-OG comprised more homogenous spindle-shaped cells. We have characterized DTSCs as STRO-1(+)/CD146(+)/CD34(+)/CD45(-) cells. However, the percentage of STRO-1(+) and CD34(+) cells was higher in DTSCs-ED (STRO-1, 17.01 ± 5.04%; CD34, 19.79 ± 4.66%) compared to DTSCs-OG cultures (STRO-1, 5.18 ± 2.39%; CD34, 9.94 ± 3.41%), probably as a result of a higher release of stem/progenitor cells from the perivascular niche during enzymatic dissociation. DTSCs isolated using either method displayed an active potential for cellular migration and biomineralization, giving rise to 3D mineralized structures when challenged with dexamethasone, monopotassium phosphate, and ß-glycerophosphate. These cellular aggregates progressively expressed differentiation markers of functional odontoblasts, including dentin sialophosphoprotein, bone sialoprotein, osteocalcin, and alkaline phosphatase, having the characteristics of osteodentin. However, in DTSCs-ED, the mineralization rate and the amount of mineralized matrix produced was higher compared to DTSCs-OG cultures. Therefore, DTSCs-ED cells display enhanced biomineralization potential, which might be of advantage for application in clinical therapy.

Push-out strength of fiber posts depending on the type of root canal filling and resin cement.

The purpose of this study was to analyze the push-out strength of two fiber post systems/resin cements (RelyX Unicem/RelyX Fiber Post (RLX) and Variolink II/DT Light SL (VL)) depending on the root canal filling (RF). One hundred sixty extracted human teeth were divided into four groups: gutta-percha/AH Plus (GP), gutta-percha/Guttaflow (GF), pre-existing root canal filling (PRF), and without root canal filling (WRF). After root canal treatment, fiber posts were inserted using either RelyX® or Variolink II®/Excite DSC®. Half of the specimens were thermocycled (TC, 5,000 cycles, 5-55°C). All specimens were subjected to the push-out test (crosshead speed 1 mm/min). Three-way ANOVA showed a significant influence of either the RF or the resin cement/post system (p < 0.001). The highest bond strength was measured for VL-WRF without TC (16.5 ± 6.4 MPa). TC had no significant influence within the RLX groups. For groups PRF and WRF, significant differences were documented between VL and RLX (PRF 16.3 ± 6.0 vs 7.0 ± 2.4 MPa, p = 0.001; WRF 16.5 ± 6.4 vs 8.0 ± 5.0, p = 0.004) before TC. No differences were found after TC. The fracture mode analysis for VL showed mainly adhesive fractures between post and cement. For RLX, mixed fractures between post and tooth and between tooth and cement were predominantly determined. The adhesion of resin cements/post systems could be dependent on the type of RF. Higher bond strength values were found for the conventional ("etch and rinse") adhesive than for the "self-adhesive resin cement."

Conservative treatment of periodontal recessions with class V-defects using gingiva-shaded composite--A systematic treatment concept.

UNLABELLED: Periodontal recessions can cause aesthetic and functional problems, especially in the anterior region or when combined with exposed crown margins. A combination of periodontal disease, recession with exposed root surface, hard-tissue defects and age emphasizes the need for treating these defects. If crown margins are exposed and surgical treatment is not possible, aesthetics and function can only be improved by replacement of the restoration. The restorative treatment option with a gingiva-shaded composite is especially valuable for dental fear patients or older patients with general or local risk factors, surgical contra-indications or Class III and IV recessions with questionable prognosis of surgery. The step-by-step-approach described in this article is an alternative, minimal-invasive treatment concept for cervical lesions in combination with all kinds of periodontal recessions, that is especially suitable for wedge-shaped defects next to exposed crown margins. CLINICAL RELEVANCE: With this treatment concept, the reader should be able to use gingiva-shaded composite for different indications, such as exposed root surfaces or crown margins in combination with recessions.

Influence of 2-hydroxyethyl methacrylate (HEMA) exposure on angiogenic differentiation of dental pulp stem cells (DPSCs).

OBJECTIVE: The angiogenic differentiation of dental pulp stem cells (DPSCs) is important for tissue homeostasis and wound healing. In this study the influence of 2-hydroxyethyl methacrylate (HEMA) on angiogenic differentiation was investigated. METHODS: To evaluate HEMA effects on angiogenic differentiation, DPSCs were cultivated in angiogenic differentiation medium (ADM) in the presence or absence of non-toxic HEMA concentrations (0.1 mM and 0.5 mM). Subsequently, angiogenic differentiation was analyzed on the molecular level by qRT-PCR and protein profiler analyzes of angiogenic markers and flow cytometry of PECAM1. The influence of HEMA on angiogenic phenotypes was analyzed by cell migration and sprouting assays. RESULTS: Treatment with 0.5 mM HEMA during differentiation can lead to a slight reduction of angiogenic markers on mRNA level. HEMA also seems to slightly reduce the quantity of angiogenic cytokines (not significant). However, these HEMA concentrations have no detectable influence on cell migration, the abundance of PECAM1 and the formation of capillaries. Higher concentrations caused primary cytotoxic effects in angiogenic differentiation experiments conducted for longer periods than 72 h. SIGNIFICANCE: Non-cytotoxic HEMA concentrations seem to have a minor impact on the expression of angiogenic markers, essentially on the mRNA level, without affecting the angiogenic differentiation process itself on a detectable level.

Camphorquinone alters the expression of extracellular proteases in a 3D co-culture model of the oral mucosa.

OBJECTIVE: Objective of our investigation was to determine the influence of CQ on the expression of antioxidant proteins and extracellular proteases in a 3D co-culture model (3DCCM) of the oral mucosa and to analyze the distribution and stability of CQ within 3D-CCMs. METHODS: 3D-CCMs consist of confluent keratinocytes (OKF6/TERT2) on cell culture inserts on top of human gingival fibroblasts (HGFs) in collagen. The treatment was carried out by adding CQ to the cell culture inserts at two time points with declining concentrations. Mass spectrometry was used to analyze the CQ concentration above and underneath the OKF6/TERT2-layer. The expression of antioxidant genes was analyzed by qRT-PCR and western blot. The regulation of extracellular proteases from different families was analyzed by qRT-PCR and Proteome Profiler arrays. RESULTS: GC/MS analysis showed that CQ was evenly distributed within the model. Heme oxygenase-1, NAD(P)H quinone dehydrogenase 1 (NQO1), and superoxide dismutase 1 were induced on the mRNA and protein level in OKF6/TERT2 cells. In HGFs, only the transcription of NQO1 was induced. The transcription of extracellular proteases was increased mainly in OKF6/TERT2 cells 72 h after the initial treatment. The quantity of ten out of 25 analyzed extracellular proteases in the cell culture supernatant above and six underneath the keratinocyte-layer were modulated by CQ. SIGNIFICANCE: Despite its high reactivity, CQ is able to penetrate a dense keratinocyte-layer, presumably across plasma membranes. CQ initially induced the cellular defense machinery against oxidative stress and altered the expression of extracellular proteases. We assume a relationship between both processes.

Evaluation of stemness properties of cells derived from granulation tissue of peri-implantitis lesions.

OBJECTIVES: Peri-implantitis (PI) is an inflammatory disease associated with peri-implant bone loss and impaired healing potential. There is limited evidence about the presence of mesenchymal stromal cells (MSCs) and their regenerative properties within the granulation tissue (GT) of infrabony peri-implantitis defects. The aim of the present study was to characterize the cells derived from the GT of infrabony PI lesions (peri-implantitis derived mesenchymal stromal cells-PIMSCs). MATERIAL AND METHODS: PIMSC cultures were established from GT harvested from PI lesions with a pocket probing depth ≥6 mm, bleeding on probing/suppuration, and radiographic evidence of an infrabony component from four systemically healthy individuals. Cultures were analyzed for embryonic (SSEA4, NANOG, SOX2, OCT4A), mesenchymal (CD90, CD73, CD105, CD146, STRO1) and hematopoietic (CD34, CD45) stem cell markers using flow cytometry. PIMSC cultures were induced for neurogenic, angiogenic and osteogenic differentiation by respective media. Cultures were analyzed for morphological changes and mineralization potential (Alizarin Red S method). Gene expression of neurogenic (NEFL, NCAM1, TUBB3, ENO2), angiogenic (VEGFR1, VEGFR2, PECAM1) and osteogenic (ALPL, BGLAP, BMP2, RUNX2) markers was determined by quantitative RT-PCR. RESULTS: PIMSC cultures demonstrated high expression of embryonic and mesenchymal stem cell markers with inter-individual variability. After exposure to neurogenic, angiogenic and osteogenic conditions, PIMSCs showed pronounced tri-lineage differentiation potential, as evidenced by their morphology and expression of respective markers. High mineralization potential was observed. CONCLUSIONS: This study provides evidence that MSC-like populations reside within the GT of PI lesions and exhibit a multilineage differentiation potential. Further studies are needed to specify the biological role of these cells in the healing processes of inflamed PI tissues and to provide indications for their potential use in regenerative therapies.

An early oral health care program starting during pregnancy: results of a prospective clinical long-term study.

This study covers phase IV of a prospective clinical long-term study. Objective of this clinical investigation was to analyze the effects of a long-term prevention program on dental and oral health of teenagers at the age of 13 to 14 years. The entire study was subdivided into four phases. Phase I comprised an individual preventive care during pregnancy ("primary-primary prevention"); phase II assessed mothers and their young children until the age of 3 years ("primary prevention"); and in phase III, mothers and children at the age of 6 years were investigated. In phase IV of the study, the oral health of 13- to 14-year-old teenagers was examined (13.4 +/- 0.5 years; n = 29). All phases consisted of an examination, education about oral health care, and treatment based on the concept of an early oral health care promotion. The control group consisted of randomly selected adolescents at the same age (n = 30). The following clinical parameters were assessed: decayed/missing/filled teeth (DMF-T)/decayed, missing, and filled surface teeth index, hygiene index, papilla bleeding index, Periodontal Screening Index, and Streptococcus mutans/Lactobacillus concentration in saliva. The teenagers of the "prevention" group of phase IV of our prospective study revealed a share of 89.7% caries-free dentitions (65.5% sound; 24.2% caries-free with fillings). Mean DMF-T was 0.55 +/- 1.0. The control group showed a significantly higher mean DMF-T of 1.5 +/- 1.5 (p < 0.05) and revealed 56.7% of caries-free dentitions (30% sound, 26.7% caries-free with restorations). Our data clearly document that an early oral health care promotion starting during pregnancy may cause a sustained and long-term improvement of the oral health of children.

The application of silicon and silicates in dentistry: a review.

Silicates and silicate-based compounds are frequently used materials in dentistry. One of their major applications is their use as fillers in different dental filling materials such as glass-ionomer cements, compomers, composites, and adhesive systems. In these materials, the fillers react with acids during the setting process or they improve the mechanical properties by increasing physical resistance, thermal expansion coefficient and radiopacity in acrylic filling materials. They also reduce polymerization shrinkage, and increase esthetics as well as handling properties. Furthermore, silicates are used for the tribochemical silication of different surfaces such as ceramics or alloys. The silicate layer formed in this process is the chemical basis for silanes that form a bond between this layer and the organic composite matrix. It also provides a micromechanical bond between the surface of the material and the composite matrix. Silicates are also a component of dental ceramics, which are frequently used in dentistry, for instance for veneers, inlays, and onlays, for denture teeth, and for full-ceramic crowns or as crown veneering materials.

HEMA modulates the transcription of genes related to oxidative defense, inflammatory response and organization of the ECM in human oral cells.

OBJECTIVES: 2-Hydroxyethyl methacrylate (HEMA) is a widely used monomer of dental resin composite materials. Incomplete curing of resins leads to elution of HEMA, which may come in contact with different cells in oral tissues. We aimed to analyze the impact of HEMA on the transcription of genes participating in detoxification of oxidative stress, inflammatory response and organization of the extracellular matrix (ECM) using human gingival fibroblasts (HGFs) and human oral keratinocytes (OKF6/TERT2). METHODS: Cells were grown in monolayer cultures and treated with different HEMA concentrations (0.5-10mM). H33342 and LDH assays were used to determine HEMA-caused cytotoxicity. Quantitative RT-PCR was used to analyze mRNA expression of four genes related to oxidative stress and five genes each related to inflammation and organization of the ECM. RESULTS: HEMA caused similar concentration-dependent cytotoxicity in fibroblasts and keratinocytes. Analysis of the transcription showed that genes were regulated in both cell types after HEMA treatment. Genes related to defense against oxidative stress were transcriptionally induced, genes related to inflammation were mainly reduced and genes related to the organization of the ECM were differentially modulated. SIGNIFICANCE: We analyzed concurrent and HEMA-dependent differential expression of 14 important genes, which have a special significance for cellular processes that are linked to redox and tissue homeostasis. The results suggest that HEMA has an impact on cellular redox-homeostasis with potential impairment of inflammatory responses and of the organization of the ECM in human gingival fibroblasts and oral keratinocytes as first target cells of eluted HEMA.

Impaired angiogenic differentiation of dental pulp stem cells during exposure to the resinous monomer triethylene glycol dimethacrylate.

OBJECTIVE: Dental pulp stem cells (DPSCs) can differentiate into tissue specific lineages to support dental pulp regeneration after injuries. Triethylene glycol dimethacrylate (TEGDMA) is a widely used co-monomer in restorative dentistry with adverse effects on cellular metabolism. Aim of this study was to analyze the impact of TEGDMA on the angiogenic differentiation potential of DPSCs. METHODS: DPSCs were characterized by flow cytometry. Short-term (max. 72h) cytotoxicity of TEGDMA was assessed by MTT assay. To evaluate TEGDMA effects on angiogenic differentiation, DPSCs were cultivated in angiogenic differentiation medium (ADM) in the presence or absence of short-term non-toxic TEGDMA concentrations (0.1mM and 0.25mM). Subsequently, angiogenic differentiation was analyzed by qRT-PCR analysis of mRNA markers and in vitro spheroid sprouting assays. RESULTS: DPSCs treated with 0.25mM TEGDMA revealed downregulation of angiogenesis-related marker genes PECAM1 (max. 3.8-fold), VEGF-A (max. 2.4-fold) and FLT1 (max. 2.9-fold) compared to respective untreated control. In addition, a reduction of the sprouting potential of DPSCs cultured in the presence of 0.25mM TEGDMA was detectable. Larger spheroidal structures were detectable in the untreated control in comparison to cells treated with 0.25mM TEGDMA. In contrast, TEGDMA at 0.1mM was not affecting angiogenic potential in the investigated time period (up to 28 days). SIGNIFICANCE: The results of the present study show that TEGDMA concentration dependently impair the angiogenic differentiation potential of DPSCs and may affect wound healing and the formation of granulation tissue.

Effects of HEMA on Nrf2-related gene expression using a newly developed 3D co-culture model of the oral mucosa.

OBJECTIVE: 2-Hydroxyethyl methacrylate (HEMA) is a component of many resin-modified materials and elutes from dental restorations into the oral cavity. Objective of our investigation was to determine the impact of HEMA on oral keratinocytes (OKF6/TERT2) and gingival fibroblasts (HGFs) in a newly established 3D co-culture model (3D-CCM) and to analyze the permeability of OKF6/TERT2 cells for HEMA. METHODS: Well-characterized 3D-CCMs, consisting of confluent OKF6/TERT2 cells on cell culture inserts above HGF-containing collagen gels, were treated supra-epithelial with HEMA. Mass spectrometry was used to measure the supra- and sub-epithelial distribution of HEMA after 24 h. The impact of HEMA on nuclear factor erythroid 2-related factor 2 (Nrf2) target genes was measured by qRT-PCR and western blot analysis. RESULTS: Mass spectrometry showed that HEMA was evenly distributed above and below the keratinocyte layer after 24 h. Analyzed target genes of Nrf2 were induced in both cell types on the mRNA-level but less pronounced in HGFs. On the protein-level, both cell types showed similar effects: At 5 mM HEMA, heme oxygenase-1 was induced 5.1-fold in OKF6/TERT2 cells and 4.1-fold in HGFs. NAD(P)H quinone dehydrogenase-1 was approximately induced 1.85-fold in both cell types. SIGNIFICANCE: Our 3D-CCM is suitable to analyze the biocompatibility of dental materials due to an improved simulation of the oral mucosa compared to monolayer cultures. Our results indicate that HEMA is able to penetrate a dense layer of keratinocytes and to activate the cellular oxidative defense response. This may be due to the activation of the Nrf2-pathway in both cell types.

Bioorganic/inorganic hybrid composition of sponge spicules: matrix of the giant spicules and of the comitalia of the deep sea hexactinellid Monorhaphis.

The giant basal spicules of the siliceous sponges Monorhaphis chuni and Monorhaphis intermedia (Hexactinellida) represent the largest biosilica structures on earth (up to 3m long). Here we describe the construction (lamellar organization) of these spicules and of the comitalia and highlight their organic matrix in order to understand their mechanical properties. The spicules display three distinct regions built of biosilica: (i) the outer lamellar zone (radius: >300 microm), (ii) the bulky axial cylinder (radius: <75 microm), and (iii) the central axial canal (diameter: <2 microm) with its organic axial filament. The spicules are loosely covered with a collagen net which is regularly perforated by 7-10 microm large holes; the net can be silicified. The silica layers forming the lamellar zone are approximately 5 microm thick; the central axial cylinder appears to be composed of almost solid silica which becomes porous after etching with hydrofluoric acid (HF). Dissolution of a complete spicule discloses its complex structure with distinct lamellae in the outer zone (lamellar coating) and a more resistant central part (axial barrel). Rapidly after the release of the organic coating from the lamellar zone the protein layers disintegrate to form irregular clumps/aggregates. In contrast, the proteinaceous axial barrel, hidden in the siliceous axial cylinder, is set up by rope-like filaments. Biochemical analysis revealed that the (dominant) molecule of the lamellar coating is a 27-kDa protein which displays catalytic, proteolytic activity. High resolution electron microscopic analysis showed that this protein is arranged within the lamellae and stabilizes these surfaces by palisade-like pillars. The mechanical behavior of the spicules was analyzed by a 3-point bending assay, coupled with scanning electron microscopy. The load-extension curve of the spicule shows a biphasic breakage/cracking pattern. The outer lamellar zone cracks in several distinct steps showing high resistance in concert with comparably low elasticity, while the axial cylinder breaks with high elasticity and lower stiffness. The complex bioorganic/inorganic hybrid composition and structure of the Monorhaphis spicules might provide the blueprint for the synthesis of bio-inspired material, with unusual mechanical properties (strength, stiffness) without losing the exceptional properties of optical transmission.

Shear bond strength of self-etch adhesives to enamel with additional phosphoric acid etching.

This study evaluated the shear bond strength of self-etch adhesives to enamel and the effect of additional phosphoric acid etching. Seventy sound human molars were randomly divided into three test groups and one control group. The enamel surfaces of the control group (n=10) were treated with Syntac Classic (SC). Each test group was subdivided into two groups (each n=10). In half of each test group, ground enamel surfaces were coated with the self-etch adhesives AdheSe (ADH), Xeno III (XE) or Futurabond NR (FNR). In the remaining half of each test group, an additional phosphoric acid etching of the enamel surface was performed prior to applying the adhesives. The shear bond strength was measured with a universal testing machine at a crosshead speed of 1 mm/minute after storing the samples in distilled water at 37 degrees C for 24 hours. Fracture modes were determined by SEM examination. For statistical analysis, one-way ANOVA and the two-sided Dunnett Test were used (p>0.05). Additional phosphoric etching significantly increased the shear bond strength of all the examined self-etch adhesives (p<0.001). The highest shear bond strength was found for FNR after phosphoric acid etching. Without phosphoric acid etching, only FNR showed no significant differences compared to the control (SC). SEM evaluations showed mostly adhesive fractures. For all the self-etch adhesives, a slight increase in mixed fractures occurred after conditioning with phosphoric acid. An additional phosphoric acid etching of enamel should be considered when using self-etch adhesives. More clinical studies are needed to evaluate the long-term success of the examined adhesives.