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.

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.

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.

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.

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.

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.

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.

Cytotoxic and genotoxic potential of the type I photoinitiators BAPO and TPO on human oral keratinocytes and V79 fibroblasts.

OBJECTIVES: Phenylbis(acyl) phosphine oxide (BAPO) and diphenyl(acyl) phosphine oxide (TPO) are alternative photoinitiators to camphorquinone (CQ) in dental resinous materials. Aim of this study was to investigate their cytotoxic/genotoxic potential in human oral keratinocytes (OKF6/Tert2) and Chinese hamster lung fibroblasts (V79) in comparison to CQ. METHODS: Cells were exposed to different concentrations of BAPO and TPO (1-50µM). Cytotoxicity was evaluated using H33342 and MTT assay, cell proliferation by BrdU proliferation assay and microscopy. Effects on cellular redox homeostasis were assessed by detecting intracellular levels of reactive oxygen/nitrogen species (ROS/RNS) using the DCFH2 assay and by quantification of mRNA expression of oxidatively regulated, cyto-protective enzymes. Genotoxic potential was determined by use of micronucleus (MN) assay. RESULTS: BAPO and TPO induced a concentration-dependent decrease of cell number. BAPO and TPO showed 50- to 250-fold higher cytotoxicity than CQ. In contrast to CQ, both photoinitiators revealed no increase of intracellular ROS/RNS. However, BAPO (10µM) at least significantly induced mRNA-expression of redox-regulated proteins after 24h similar to 2.5mM CQ. Additionally, BAPO significantly raised the number of micronuclei, but only in V79 cells (10µM: 12±1, 2.5mM CQ: 15±1, medium control: 6±3). However, it also significantly decreased proliferation of these cells (10µM BAPO: 19.8%±7.3% compared to controls). SIGNIFICANCE: BAPO and TPO revealed concentration-dependent cytotoxic effects in human oral keratinocytes and V79 cells. However, in contrast to CQ, no generation of intracellular ROS/RNS was found. Only BAPO induced genotoxicity in V79 cells.

Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects "stemness" properties.

BACKGROUND: Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term "stemness" of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion. METHODS: DPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (ß-galactosidase (SA-ß-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR. RESULTS: Similar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6-7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in "osteogenic pre-disposition", evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6-7 under all expansion conditions. CONCLUSIONS: These findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.

Human treated dentin matrices combined with Zn-doped, Mg-based bioceramic scaffolds and human dental pulp stem cells towards targeted dentin regeneration.

OBJECTIVE: This study aimed to investigate the potential of Mg-based bioceramic scaffolds combined with human treated-dentin matrices (hTDMs) and dentinogenesis-related morphogens to promote odontogenic differentiation and dentin-like tissue formation by Dental Pulp Stem Cells-DPSCs. METHODS: DPSC cultures were established and characterized by flow cytometry. Experimental cavities were prepared inside crowns of extracted teeth and demineralized by EDTA (hTDMs). Zn-doped, Mg-based bioceramic scaffolds, synthesized by the sol-gel technique, were hosted inside the hTDMs. DPSCs were spotted inside the hTDMs/scaffold constructs with/without additional exposure to DMP-1 or BMP-2 (100ng/ml, 24h). Scanning Electron Microscopy-SEM, live/dead fluorescence staining and MTT assay were used to evaluate cell attachment and viability; Real time PCR for expression of osteo/odontogenic markers; Inductively Coupled Plasma-Atomic Emission Spectrometry-ICP/AES for scaffold elemental release analysis; ELISA for hTDM growth factor release analysis; SEM and X-ray Diffraction-XRD for structural/chemical characterization of the regenerated tissues. RESULTS: Scaffolds constantly released low concentrations of Mg(2+), Ca(2+), Zn(2+) and Si(4+), while hTDMs growth factors, like DMP-1, BMP-2 and TGFß-1. hTDMs/scaffold constructs supported DPSC viability, inducing their rapid odontogenic shift, indicated by upregulation of DSPP, BMP-2, osteocalcin and osterix expression. Newly-formed Ca-P tissue overspread the scaffolds partially transforming into bioapatite. Exposure to DMP-1 or BMP-2 pronouncedly enhanced odontogenic differentiation phenomena. SIGNIFICANCE: This is the first study to validate that combining the bioactivity and ion releasing properties of bioceramic materials with growth factor release by treated natural dentin further supported by exogenous addition of key dentinogenesis-related morphogens (DMP-1, BMP-2) can be a promising strategy for targeted dentin regeneration.

A biomimetic approach to ameliorate dental hypersensitivity by amorphous polyphosphate microparticles.

OBJECTIVE: Dental hypersensitivity has become one of the most common and most costly diseases in the world, even though those maladies are very rarely life threatening. Using amorphous microparticles, fabricated from the natural polymer (polyphosphate), we intend to reseal the dentinal tubules exposed and reduce by that the hypersensitivity. METHODS: Amorphous microparticles (termed aCa-polyP-MP) were prepared from Na-polyphosphate (polyP) and CaCl2, then incubated with human teeth. The potential of the microparticles to plug the dentinal tubules was determined by microscopic and spectroscopic techniques. RESULTS: We demonstrate that, in contrast to polyP, the aCa-polyP-MP efficiently reseal dentinal tubules exposed at the tooth surface. Scanning electron microscopical (SEM) and energy dispersive X-ray spectroscopic (EDX) studies showed that the tooth cement and dentin surfaces, incubated with aCa-polyP-MP, form a nearly homogenous, approximately 50-µm thick solid polyP layer on the tooth cement and dentin surfaces, while no coating on the tooth surface, incubated with Na-polyP [Ca(2+)], was observed. Determination of the mechanical properties of the polyP coating revealed a Martens hardness of 3.85±0.64GPa and a reduced elastic modulus of 94.72±8.54GPa already after a 3h exposure to the aCa-polyP-MP, which become close to those of the natural enamel (4.33±0.69GPa and 101.61±8.52GPa, respectively) after prolonged incubation periods. In addition, aCa-polyP-MP turned out to display morphogenetic activity. Incubation of precursor odontoblasts cultures in the presence of aCa-polyP-MP resulted in a 7-fold increase of the steady-state-expression level of the gene encoding for the alkaline phosphatase (ALP) during a 7 d incubation period. SIGNIFICANCE: Ca-polyP microparticles, consisting of the biocompatible natural polymer polyP, provide a potential sealing material for dentinal tubules on the tooth surface.

Curcumin in Combination with Piperine Suppresses Osteoclastogenesis In Vitro.

INTRODUCTION: The dietary pigment curcumin is a natural polyphenol extracted from the Curcuma longa rhizomes native to South Asia. The antioxidative, antimicrobial, and anti-inflammatory activities besides its unknown side effects suggest that curcumin could be a promising antiresorptive agent to prevent replacement resorption in replanted teeth after traumatic avulsion. Piperine, an alkaloid present in black pepper, seems to enhance the bioavailability and activity of curcumin. Therefore, this study evaluated the biocompatibility of curcumin and piperine in cultures of periodontal ligament cells as well as their effects in an in vitro osteoclastogenesis model of RAW 264.7 macrophages. METHODS: The cytotoxicity in human periodontal ligament fibroblasts, human osteogenic sarcoma cells (SAOS-2), and murine osteoclastic precursors (RAW 264.7) was analyzed by using cell number determination and proliferation assays. The ability of curcumin and its conjugate to suppress the receptor activator of nuclear factor kappa B ligand-induced osteoclastogenesis was assessed by tartrate-resistant acid phosphatase (TRAP) staining and activity as well as real-time polymerase chain reaction. RESULTS: Curcumin at concentrations ≥ 10 µmol/L was cytotoxic in all cell types tested, whereas piperine showed only slight cytotoxicity at 30 µmol/L in RAW and SAOS cultures. Although curcumin caused already significant effects, the combination with piperine completely suppressed the osteoclastogenesis by decreasing the TRAP activity and inhibiting the expression of the specific osteoclast markers TRAP, cathepsin K, and calcitonin receptor. CONCLUSIONS: We demonstrated that curcumin combined with piperine suppressed the osteoclastogenesis in vitro without causing cytotoxic effects in periodontal ligament cells. These findings suggest its potential therapeutic application for the prevention and treatment of replacement resorption in replanted avulsed teeth.

Angiogenic Potential and Secretome of Human Apical Papilla Mesenchymal Stem Cells in Various Stress Microenvironments.

Stem cells from the apical papilla (SCAP) of human adult teeth are considered an accessible source of cells with angiogenic properties. The aims of this study were to investigate the endothelial transdifferentiation of SCAP, the secretion of pro- and antiangiogenic factors from SCAP, and the paracrine effects of SCAP when exposed to environmental stress to stimulate tissue damage. SCAP were exposed to serum deprivation (SD), glucose deprivation (GD), and oxygen deprivation/hypoxia (OD) conditions, individually or in combination. Endothelial transdifferentiation was evaluated by in vitro capillary-like formation assays, real-time polymerase chain reaction, western blot, and flow cytometric analyses of angiogenesis-related markers; secretome by antibody arrays and enzyme-linked immunosorbent assays (ELISA); and paracrine impact on human umbilical vein endothelial cells (HUVECs) by in vitro transwell migration and capillary-like formation assays. The short-term exposure of SCAP to glucose/oxygen deprivation (GOD) in the presence, but mainly in deprivation, of serum (SGOD) elicited a proangiogenesis effect indicated by expression of angiogenesis-related genes involved in vascular endothelial growth factor (VEGF)/VEGFR and angiopoietins/Tie pathways. This effect was unachievable under SD in normoxia, suggesting that the critical microenvironmental condition inducing rapid endothelial shift of SCAP is the combination of SGOD. Interestingly, SCAP showed high adaptability to these adverse conditions, retaining cell viability and acquiring a capillary-forming phenotype. SCAP secreted higher numbers and amounts of pro- (angiogenin, IGFBP-3, VEGF) and lower amounts of antiangiogenic factors (serpin-E1, TIMP-1, TSP-1) under SGOD compared with SOD or SD alone. Finally, secretome obtained under SGOD was most effective in inducing migration and capillary-like formation by HUVECs. These data provide new evidence on the microenvironmental factors favoring endothelial transdifferentiation of SCAP, uncovering the molecular mechanisms regulating their fate. They also validate the angiogenic properties of their secretome giving insights into preconditioning strategies enhancing their therapeutic potential.

Genotoxic effects of camphorquinone and DMT on human oral and intestinal cells.

OBJECTIVE: Released components of oral biomaterials can leach into the oral cavity and may subsequently reach the gastrointestinal tract. Camphorquinone (CQ) is the most common used photoinitiator in resinous restorative materials and is often combined with the co-initiator N,N-dimethyl-p-toluidine (DMT). It has been shown that CQ exerts cytotoxic effects, at least partially due to the generation of reactive oxygen species (ROS). Objective of this study was to examine the cytotoxic and genotoxic potential of CQ in human oral keratinocytes (OKF6/TERT2) and immortalized epithelial colorectal adenocarcinoma cells (Caco-2). Furthermore, the effects of visible-light irradiation and the co-initiator DMT were investigated as well as the generation of ROS, the potential protective effect of glutathione (GSH) and a recovery period of CQ-treated Caco-2 cells. METHODS: The alkaline comet assay was used to determine DNA damage. Additionally, an enzyme modified comet assay was applied, which detects 7,8-dihydro-8-oxoguanine (8-oxoguanine), a reliable marker for oxidative stress. RESULTS: Our data revealed that high concentrations of CQ induced DNA lesions in OKF6/TERT2 cells. This DNA damage is at least partly caused by the generation of 8-oxoguanine. In addition, CQ and DMT increased ROS formation and induced DNA damage in Caco-2 cells. CQ-treatment resulted in generation of 8-oxoguanine. The antioxidant GSH efficiently prevented CQ-associated DNA damage. Furthermore, a recovery following CQ-treatment significantly reduced DNA damage. SIGNIFICANCE: We conclude that CQ-induced DNA damage is caused by oxidative stress in oral and intestinal cells. These lesions can be prevented and possibly repaired by GSH-treatment and recovery of cells after the photoinitiator is removed from cultures.

Wnt/ß-catenin signaling regulates Dental Pulp Stem Cells' responses to pulp injury by resinous monomers.

OBJECTIVES: Aim of this study was to investigate whether Dental Pulp Stem Cells-DPSCs responses to pulp injury caused by resinous monomers is be mediated through activation of Wnt/ß-catenin signaling. METHODS: DPSCs cultures were established from third molars of healthy donors and characterized for stem cell markers with flow cytometry. Cells were exposed to TEGDMA (T: 0.5-2mM) with or without presence of the Wnt-1 ligand (W:25-100ng/ml) or the GSK3ß inhibitor Lithium (L:1-10mM), used both as activators of Wnt/ß-catenin signaling. Cell viability was evaluated by MTT assay, cell cycle profiles by flow cytometry and expression of key molecules of Wnt/ß-catenin signaling by Real-time PCR and Western Blot. RESULTS: DPSC exposure to TEGDMA caused a concentration-dependent cytotoxicity, accompanied by G1 arrest at lower and G2/M arrest at higher concentrations or after prolonged exposure. Lithium caused a dual effect, by stimulating/inhibiting cell proliferation at lower/higher concentrations respectively and causing a G2/M arrest in a concentration-dependent manner. Wnt signaling could be activated in DPSCs after Lithium or Wnt-1 treatment, as shown by accumulation of ß-catenin, its translocation into the nucleus and enhanced expression of key pathway players, like LEF1 and Cyclin D1. Importantly, exposure to TEGDMA caused a more pronounced activation of the pathway, whereas cumulative effects were observed after T/L or T/W co-treatment, indicating a very strong activation of Wnt signaling after treatment of already "activated" (by Lithium or Wnt-1) cells with TEGDMA. SIGNIFICANCE: These findings highlight the important role of Wnt canonical signaling in pulp repair responses to common injuries.

Release and protein binding of components from resin based composites in native saliva and other extraction media.

OBJECTIVES: Unpolymerized (co)monomers and additives can be released from resin based composites (RBCs) and can enter the human organism. In this study, the binding of ingredients from composites to salivary proteins and plasma proteins was investigated. METHODS: The composites investigated were Admira(®) flow, Venus(®) Diamond flow, Filtek™ Supreme XTE flow, Tetric EvoCeram(®), Tetric EvoFlow(®). The samples (n=4) were polymerized according to the instructions of the manufacturer of RBCs. The samples were immersed into native saliva, protein-free saliva (artificial saliva), water and ethyl acetate, and incubated at 37°C for 24h or 72h. The eluates were analyzed by gas chromatography/mass spectrometry. To determine the binding to salivary proteins, the concentration of (co)monomers and additives detected in native saliva was compared to the concentration of (co)monomers and additives detected in protein-free saliva, water and ethyl acetate respectively. To assess the affinity of TEGDMA, EGDMA, DEGDMA, PMGDMA, BPA, and DCHP to human serum albumin (HSA) and human α1-acid glycoprotein (AGP), a plasma protein binding assay (ABNOVA, Transil XL PPB Prediction Kit TMP-0212-2096) was performed. The statistical significance (p<0.05) of the difference between the experimental groups was tested using the one-way-analysis of variance (ANOVA), followed by Tukey's analysis. RESULTS: The concentration of TEGDMA, GMA and CyHEMA released in native saliva was significantly lower than the concentration released in protein-free saliva or water (Admira(®) flow: concentration of TEGDMA after 72h: 0.08 mmol/L (native saliva), 0.34 mmol/L (protein-free saliva), 0.39 mmol/L (water)). The concentrations of HEMA, EGDMA, DDDMA and CQ released in native saliva remained even below the detection limit, compared to the other extraction media. Protein binding of the tested methacrylates to HSA+AGP was 82-85%, the binding of DCHP was 96.6%, and the binding of BPA was 95.2%. SIGNIFICANCE: Artificial saliva or water as extraction medium does not reflect the real physiological situation in the body. Salivary and plasma proteins may bind (co)monomers and additives and may thereby contribute to a lower bioavailability of leachables from RBCs in vivo than previously thought.

Effects of alendronate on osteoclast formation and activity in vitro.

INTRODUCTION: Root resorption is a common complication after replantation following traumatic dental avulsion. Endodontic therapy combined with local and intracanal medications aims to avoid osteoclastic activity. In such cases, the application of alendronate (ALN), a bisphosphonate widely used for the treatment of bone disorders, could be of clinical relevance. This study evaluated alendronate biocompatibility on periodontal ligament cells as well as its effects on an in vitro osteoclastogenesis model. METHODS: Alendronate cytotoxicity (10(-3) to 10(-9) mol/L) in human periodontal ligament fibroblasts, human osteogenic sarcoma cells, and murine osteoclastic precursors (RAW 264.7) was analyzed using cell number determination, cell viability, and proliferation assays. ALN (10(-6) to 10(-12) mol/L) effects on RANKL-induced osteoclastogenesis of RAW cells were assessed by tartrate-resistant acid phosphatase (TRAP) staining and activity and real-time polymerase chain reaction. RESULTS: ALN at higher concentrations was cytotoxic for all cell types, inhibiting significantly the proliferation of human osteogenic sarcoma cells and human periodontal ligament fibroblasts (≥10(-5) mol/L). TRAP activity and expression of the osteoclast markers TRAP and cathepsin K by RAW-derived osteoclasts decreased significantly with ALN at low concentrations, reaching the maximum effect at 10(-10) mol/L. CONCLUSIONS: We showed that ALN at very low concentrations is an effective inhibitor of RANKL-generated osteoclasts, without causing cytotoxic effects on their precursors or periapical cells. ALN at such concentrations might be useful to prevent replacement resorption in avulsed teeth.

Dental pulp stem cells' secretome enhances pulp repair processes and compensates TEGDMA-induced cytotoxicity.

OBJECTIVES: Aim of this study was to investigate the effects of dental pulp stem cells' (DPSCs) secretome, expressed through their culture conditioned medium (CM), on biological endpoints related to pulp repair and on TEGDMA-induced cytotoxicity. METHODS: DPSCs cultures were established and characterized for stem cell markers with flow cytometry. CM was collected from DPSCs under serum deprivation conditions (SDC) and normal serum conditions (NSC) at various time-points. CM effects on DPSCs viability, migration and mineralization potential were evaluated by MTT assay, transwell insert and in vitro scratch assay and Alizarin Red staining/quantification respectively. TEGDMA (0.25-2.0mM) cytotoxicity regarding the same biological endpoints was tested in the presence/absence of CM. TGF-ß1 and FGF-2 secretion in CM was measured by ELISA. RESULTS: CM collected under SDC (4d) was able to increase cell viability by 20-25% and to reduce TEGDMA cytotoxicity by 20% (p<0.05). CM positive effects were not obvious when collected under NSC. Transwell assay showed significant increase (26%, p<0.05) of DPSCs' migration after CM exposure, whereas both migration assays could not support a migration rate improvement in TEGDMA-treated cultures exposed to CM compared to TEGDMA alone. CM significantly (p<0.01) increased DPSCs mineralization potential and completely counteracted TEGDMA cytotoxicity on this process. ELISA analysis showed a time-dependent increase of TGF-ß1 and a TEGDMA concentration-dependent increase of both TGF-ß1 and FGF-2 in CM. SIGNIFICANCE: These findings suggest that DPSCs secretome increases their viability, migration and mineralization potential and counteracts TEGDMA-induced cytotoxicy, revealing a novel mechanism of DPSCs autocrine signaling on pulp repair processes.