Cervical Metastases Behavior of T1-2 Squamous Cell Carcinoma of the Tongue.

INTRODUCTION: The cervical lymph node metastasis (CM) is one of the most important prognostic factors for oral squamous cell carcinoma. Although the frequency and distribution of CM for tongue carcinoma (TC) are well documented in the literature, there is only little data on metastasis patterns depending on the location of the cancer within the tongue. MATERIALS AND METHODS: In a retrospective study all patients with a T1-T2 TC who were treated between 1997 and 2013 were analysed regarding epidemiological data, risk factors, and tumour parameters such as exact localization, CM. RESULTS: 204 patients (59 ± 15 years; ♀37 %, ♂63 %) were included. At the initial diagnosis 23 % had an advanced tumour stage (III-IV) due to CM. The occurrence of CM was significantly higher for T2, advanced G-status and the localization in the posterior area of the tongue. The presence of CM-but not the recurrence-had a significant influence on the survival rate. CONCLUSION: The small TC has an aggressive metastatic behaviour depending not only on the classical prognostic factors such as grading and tumour size, but is also strongly influenced by the posterior location within the tongue.

The effect of different collagen modifications for titanium and titanium nitrite surfaces on functions of gingival fibroblasts.

OBJECTIVES: Targeted modifications of the bulk implant surfaces using bioactive agents provide a promising tool for improvement of the long-term bony and soft tissue integration of dental implants. In this study, we assessed the cellular responses of primary human gingival fibroblasts (HGF) to different surface modifications of titanium (Ti) and titanium nitride (TiN) alloys with type I collagen or cyclic-RGDfK-peptide in order to define a modification improving long-term implants in dental medicine. MATERIALS AND METHODS: Employing Ti and TiN implants, we compared the performance of simple dip coating and anodic immobilization of type I collagen that provided collagen layers of two different thicknesses. HGF were seeded on the different coated implants, and adhesion, proliferation, and gene expression were analyzed. RESULTS: Although there were no strong differences in initial cell adhesion between the groups at 2 and 4 hours, we found that all surface modifications induced higher proliferation rates as compared to the unmodified controls. Consistently, gene expression levels of cell adhesion markers (focal adhesion kinase (FAK), integrin beta1, and vinculin), cell differentiation markers (FGFR1, TGFb-R1), extracellular protein markers (type I collagen, vimentin), and cytoskeletal protein marker aktinin-1 were consistently higher in all surface modification groups at two different time points of investigation as compared to the unmodified controls. CONCLUSION: Our results indicate that simple dip coating of Ti and TiN with collagen is sufficient to induce in vitro cellular responses that are comparable to those of more reliable coating methods like anodic adsorption, chemical cross-linking, or RGD coating. TiN alloys do not possess any positive or adverse effects on HGF. CLINICAL RELEVANCE: Our results demonstrate a simple, yet effective, method for collagen coating on titanium implants to improve the long term integration and stability of dental implants.

The influence of geranylgeraniol on microvessel sprouting after bisphosphonate substitution in an in vitro 3D-angiogenesis assay.

OBJECTIVES: Recent studies focused on angiogenesis in the pathophysiology of bisphosphonate-associated osteonecrosis of the jaws (BP-ONJ) and identified geranylgeraniol (GGOH) as a feasible option for BP-ONJ therapy. This study investigated the influence of GGOH on microvessel sprouting after BP-incubation in vitro. MATERIALS AND METHODS: Ten experimental set-ups were randomly designed in an in vitro 3D-angiogenesis assay. Two groups included HUVEC cell spheroids with and without (±) GGOH substitution as controls and eight groups pairwise contained either clodronate or the nitrogen-containing bisphosphonates (N-BP) ibandronate, pamidronate, and zoledronate ± GGOH. The size of the cell spheroids including the outbranching sprouts (SpS) as well as the density (SpD) and length of the sprouts (SpL) were analyzed by a grid system after 0, 24, 48, and 72 h. RESULTS: For controls and NN-BP clodronate, no significant differences at any tested parameter and any point of measurement could be detected within the experimental set-ups ± GGOH (p each ≥0.05). For N-BP ibandronate, the experimental set-ups +GGOH showed a significantly increased SpS, SpD, and SpL after 48 and 72 h (p each ≤0.002) compared to the experimental set-ups -GGOH. For N-BPs pamidronate and zoledronate, the experimental set-ups + GGOH demonstrated a significantly increased SpS, SpD, and SpL after 24, 48, and 72 h (p each ≤0.001) compared to the experimental set-ups -GGOH. CONCLUSIONS: The strong negative influence of N-BPs on microvessel sprouting could be significantly reversed by GGOH. CLINICAL RELEVANCE: Since supportive therapeutic options for BP-ONJ are lacking, GGOH might be a promising substitute for BP-ONJ prevention and therapy.

Isoprenoid geranylgeraniol: the influence on cell characteristics of endothelial progenitor cells after bisphosphonate therapy in vitro.

OBJECTIVES: Geranylgeraniol (GGOH) has been reported as a potential treatment option for bisphosphonate-associated osteonecrosis of the jaws (BP-ONJ). The aim of this study was to analyze the effects of GGOH on endothelial progenitor cells (EPC) after bisphosphonate treatment in vitro. MATERIALS AND METHODS: EPC were incubated with different nitrogen (N-BPs: ibandronate, pamidronate, zoledronate) and a non-nitrogen-containing bisphosphonates (NN-BP: clodronate) with and without GGOH. Cell viability was measured by MTT and PrestoBlue assay. Migration ability was analyzed with a Boyden and Scratch assay. Apoptosis rates were determined by colony-forming, Tunel and ToxiLight assays. RESULTS: Negative effects of N-BPs on EPC were shown in all tests without GGOH. The substitution of GGOH demonstrated significantly increased cell viability (MTT: p each N-BP ≤0.004; PrestoBlue: p each N-BP <0.001) and migration ability (Boyden: p each N-BP <0.001; Scratch: p each N-BP <0.001). Concerning the apoptosis rates, increased EPC colony densities (p each N-BP ≤0.009), decreased numbers of apoptotic cells in the Tunel assay (p each N-BP <0.001), and a decreased adenylate kinase release in the ToxiLight assay (p each N-BP ≤0.03) were observed. For the clodronate-treated cells, no significant differences could be detected with or without GGOH in any assay (p each N-BP/NN-BP >0.05). CONCLUSIONS: GGOH cell treatment reversed the negative effects of bisphosphonates on EPC. CLINICAL RELEVANCE: These findings support the hypothesis that systemic or local GGOH treatment might lead to new therapeutic strategies for BP-ONJ.

A chemiluminescent light system in combination with toluidine blue to assess suspicious oral lesions-clinical evaluation and review of the literature.

OBJECTIVES: The aim of the study was an evaluation of an acetic acid wash and chemiluminescent light system in combination with toluidine blue in order to detect visual identified, potentially malignant lesions. MATERIALS AND METHODS: Forty-four patients with 50 oral lesions of primary uncertain visible dignity were included. Next to a clinical examination, a screening with ViziLite® (VL) as well as toluidine blue (TB; together ViziLite® Plus (VLP)) was conducted. Histopathology served as gold standard and sensitivity (SE), specificity (SP), positive as well as negative predictive value (PPV, NPV) was calculated descriptively. Additionally, a PubMed literature search using the key words "ViziLite" and "chemiluminescence oral cancer" was conducted. RESULTS: Histological diagnosis showed 40 lesions of reactive/inflammatory nature, moderate dysplasia (n = 3) and oral squamous cell carcinoma (OSCC n = 7). All OSCCs and one dysplasia were identified correct via clinical diagnosis (SE 90 %, SP 100 %, PPV 100 %, NPV 97.5 %). VL examination could show all malignancies with low specificity (SE 100 %, SP 30 %, PPV 26 %, NPV 100 %). TB and VLP were positive in all cases of cancer and in one case of inflammation (SE 80 %, SP 97.5 %, PPV 89 %, NPV 95 %). In the review, eight clinical trials with similar results were included. CONCLUSIONS: The adjunct of TB to VL reduces the number of false positives without increasing the rate of false negatives. CLINICAL RELEVANCE: Clinical evidence to justify the additional cost of the system for diagnosis of suspicious lesions is weak. However, for the potential role of VLP in detection of lesions not otherwise identified in the visual exam in general dental practice further studies are required.

The prevalence of human papilloma virus (HPV) infections in oral squamous cell carcinomas: a retrospective analysis of 88 patients and literature overview.

In addition to tobacco and alcohol consumption, the two main risk factors for oral squamous cell carcinoma (OSCC), recent studies have revealed infections with human papilloma virus (HPV) as an additional risk factor for OSCC development. In the field of head and neck malignancies, the prevalence of HPV infections in oropharyngeal cancer (OC) ranges in different studies up to 84%. While HPV infection is discussed as an independent risk factor in this region, its distinguished role in carcinogenesis of tumours localized to the oral cavity remains still uncertain. In this study, we analysed the HPV status in 88 consecutive patients with OSCCs localized anterior of the palatoglossal arch who were treated in the Department of Oral and Maxillofacial Surgery at the University Medical Center Mainz. The HPV status analysis was performed using DNA-PCR and immunostaining of p16 protein. The prevalence of HPV-positive OSCCs was about 6% (5 patients). In 3 patients the HPV subtypes 16/18 were found. No significant differences between the HPV positive and negative patients regarding age, gender, smoking and alcohol consumption, localization and TNM level could be detected. Contrary to other studies focussing on cancers of the lingual and palatine tonsil, the prevalence of HPV infections was much lower in the oral cavity. Therefore HPV infection might play a less important role in oral carcinogenesis.

Influence of CAD/CAM all-ceramic materials on cell viability, migration ability and adenylate kinase release of human gingival fibroblasts and oral keratinocytes.

OBJECTIVES: The aim of this study was to analyze the influence of four CAD/CAM all-ceramic materials on cell viability, migration ability and adenylate kinase (ADK) release of human gingival fibroblasts (HGF) and oral keratinocytes (HOK). MATERIALS AND METHODS: HGF and HOK were cultured on disc-shaped CAD/CAM all-ceramic materials (e.max CAD LT, e.max CAD HT, Empress CAD and Mark II) and on discs made of tissue culture polystyrene surface (TCPS) serving as control. Cell viability was analyzed by using an MTT assay, and migration ability was investigated by a scratch assay. A ToxiLight assay has been performed to analyze the effect of all-ceramic materials on ADK release and cell apoptosis. RESULTS: At MTT assay for HGF, no significant decrease of cell viability could be detected at all points of measurement (p each > 0.05), while HOK demonstrated a significant decrease in cell viability especially on Empress CAD and Mark II at each point of measurement (p each < 0.001). Scratch assay demonstrated an increased migration ability for HGF on e.max CAD HT, Empress CAD and Mark II (p each < 0.001), whereas HOK showed a significantly decreased migration ability on all tested materials at all points of measurement (between -36 % and -71 %; p each < 0.001). At ToxiLight assay, only small cytotoxic effects of the all-ceramic materials could be investigated. CONCLUSIONS: This study disclosed significant differences in cell viability and migration ability of HGF and HOK on CAD/CAM all-ceramic materials. CLINICAL RELEVANCE: CAD/CAM all-ceramic materials can influence oral cell lines responsible for soft tissue creation which may affect the esthetic outcome.

Bisphosphonates' antiangiogenic potency in the development of bisphosphonate-associated osteonecrosis of the jaws: influence on microvessel sprouting in an in vivo 3D Matrigel assay.

OBJECTIVES: Bisphosphonate-associated osteonecrosis of the jaw (BP-ONJ) is an adverse side effect of long-term bisphosphonate treatment. One theory of BP-ONJ etiology suggests a negative influence of these agents on angiogenesis and vascularization. This in vivo study analyzed the effects of bisphosphonates on angiogenesis in a 3D Matrigel assay. MATERIALS AND METHODS: Matrigel plugs were implanted into fifty 6-8-week-old female nude mice. Ten animals each were treated either with clodronate, ibandronate, pamidronate, zoledronate, or carrier solution as controls. The microvessel density (MVD), microvessel area (MVA), and microvessel size (MVS) in Matrigel plugs were analyzed after 21 days of treatment by immunohistochemistry and exemplary 3D microvascular corrosion castings. RESULTS: All bisphosphonates induced a statistically significant decrease of MVD (p each <0.001), whereby the nitrogen-containing bisphosphonates (N-BPs) demonstrated a clearly stronger effect than non-nitrogen-containing bisphosphonates (NN-BP) clodronate (control 166, clodronate 99, ibandronate 48, pamidronate 47, zoledronate 35 microvessels/mm(2)). Referring to MVA, similar results could be detected. MVS was significantly increased especially by ibandronate (103 %) compared to control group (p < 0.001). Scanning electron microscope scans of the corrosion castings confirmed these results. CONCLUSIONS: The stronger influence on MVD by N-BPs compared to the NN-BP clodronate may explain for the lack of BP-ONJ after treatment with NN-BPs. CLINICAL RELEVANCE: Ibandronate induced a strong increase of MVS. In combination with the reduced MVD, this could result only in a fractional reduced perfusion which might be an explanation for the lower occurrence of BP-ONJ in patients receiving ibandronate compared to patients receiving pamidronate or zoledronate.

In vitro and in vivo characterization of porcine acellular dermal matrix for gingival augmentation procedures.

BACKGROUND AND OBJECTIVE: Recently, porcine acellular dermal matrix (PADM) has been proposed as a possible alternative to autogenous grafts in periodontal plastic surgery. The aim of the present study was to investigate the in vitro responses of four different oral cell lines cultured on a novel PADM. Furthermore, tissue reaction to PADM was evaluated histologically after subcutaneous implantation in mice. MATERIAL AND METHODS: Human gingival fibroblasts (HGF), human osteoblast-like cells, human umbilical vein endothelial cells and human oral keratinocytes (HOK) were cultured and transferred on to the PADM. A tissue culture polystyrene surface served as the control. The viability of all tested cell lines on PADM was measured by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetric assay and PrestoBlue(®) reagent. The ToxiLight(®) assay was performed to analyze the effect of PADM on adenylate kinase release. PADM was implanted into nude mice subcutaneously and subjected to histological analysis after 21 d. RESULTS: Using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetric assays, all tested cell lines cultured on PADM demonstrated a significant increase of viability compared to the control group (each p < 0.001) with the exception of HGF and HOK after 3 d (each p > 0.05). According to the PrestoBlue(®) analysis, all cell lines demonstrated a significant increase of viability compared to the control group at the particular points of measurement after 18 h (HGF p < 0.01; human osteoblast-like cells, human umbilical vein endothelial cells, HOK each p < 0.001). No significant cytotoxic effects of PADM on the tested cell lines could be observed, as assessed by changes in adenylate kinase release. Subcutaneous implantation of PADM into nude mice demonstrated good integration with surrounding tissues and significant revascularization of its collagen structure. CONCLUSION: Overall, the results suggest that PADM is a promising substitute for autogenous soft tissue grafts in periodontal surgery.

Geranylgeraniol - a new potential therapeutic approach to bisphosphonate associated osteonecrosis of the jaw.

Bisphosphonate associated osteonecrosis of the jaw (BP-ONJ) is one of the main side effects of bisphosphonate therapy (BPT). To date, there is no effective therapy of the BP-ONJ. Nitrogen-containing bisphosphonates (N-BPs) are particularly able to inhibit pyrophosphate synthase (FPPS) in the mevalonate pathway (MVP). Consequent of decreased synthesis of the metabolite Geranylgeraniol (GGOH) is believed to largely account for the development of BP-ONJ. Negative effect of N-BPs could be shown, resulting in decreased viability and migration capacity of different cell types of hard and soft tissues such as osteoblasts, fibroblast und endothelial cells. Aim of our in vitro study was to demonstrate that the mevalonate pathway metabolite GGOH could reverse the negative biological effect of N-BPs. Biological effect of GGOH on bisphosphonate-treated human umbilicord vein endothelial cells (HUVEC), fibroblast and osteogenic cells was analyzed by a viability test and measuring the migration capacity in a scratch wound assay as well as a migration assay using Boyden chambers. The morphological cell architecture of the treated cells was analyzed by phallacidin staining. GGOH cell-treatment can rescue the negative effect of bisphosphonates. These results underline the hypothesis that systemic or local treatment with GGOH could lead to new therapeutic strategies for BP-ONJ.

Bisphosphonates affect migration ability and cell viability of HUVEC, fibroblasts and osteoblasts in vitro.

OBJECTIVES: Bisphosphonate-associated osteonecrosis of the jaw (BP-ONJ) is a side effect in patients being treated with bisphosphonates. The bisphosphonates most often associated with BP-ONJ are the highly potent nitrogen-containing bisphosphonates, e.g. pamidronate or zoledronate. In terms of BP-ONJ aetiology, several theories are being discussed: inhibition of bone remodelling, effect on soft tissues, and antiangiogenic effect of bisphosphonates. The aim of this in vitro study was to investigate the effect of different potent bisphosphonates on osteoblasts, fibroblasts and human umbilicord vein endothelial cells (HUVEC). MATERIALS AND METHODS: Three nitrogen-containing bisphosphonates (ibandronate, pamidronate and zoledronate) and one non-nitrogen-containing bisphosphonate (clodronate) were compared concerning their potency on apoptosis induction (tunel), cell viability (calcein assay) and migration potency (boyden chamber) on osteoblasts, fibroblasts and HUVEC. RESULTS: The nitrogen-containing bisphosphonates, particularly pamidronate and zoledronate, affect cell viability, cell migration and the induction of apoptosis of osteoblasts, fibroblasts and HUVEC. CONCLUSIONS: These results support the theory that BP-ONJ is a multifactorially caused disease because several cell lines of the oral cavity which are responsible for integrity and wound healing are negatively affected by nitrogen-containing bisphosphonates. Perioperative interruption of bisphosphonate application during dental surgical procedures--if possible--might be feasible to promote better wound healing.

Influence of bisphosphonates on endothelial cells, fibroblasts, and osteogenic cells.

Bisphosphonate-associated osteonecrosis of the jaws (BP-ONJ) is a side effect primarily in patients receiving highly potent nitrogen-containing bisphosphonates. The exact etiopathology is unknown. In addition to reduced bone remodeling, there may also be an impact on soft tissues. The impact of nitrogen- (ibandronate, pamidronate, zoledronate) and non-nitrogen-containing bisphosphonates (clodronate) on human umbilicord vein endothelial cells (HUVEC), fibroblasts and osteogenic cells was analyzed employing cell viability testing and a scratch wound assay. The impact on the cell morphology of vital-stained osteogenic cells was investigated by cell visualization (confocal laser scanning microscopy). Pamidronate and zoledronate had the greatest negative impact on all cell lines, whereas the impact of ibandronate and clodronate was less distinct. The effect of clodronate on HUVEC and fibroblasts was particularly marginal. BP-ONJ could be a multifactorial event with multicellular impairments. This might result in altered wound healing. The increased impact of the highly potent bisphosphonates, particularly on non-bone cells, may explain the higher occurrence of BP-ONJ.

[Metabolit-induced bioluminescence for tumor prediction: detection of metabolites in tumors of the head and neck region]. / Metabolitinduzierte Biolumineszenz zur Tumorprädiktion: Detektion von Metaboliten in Tumoren des Kopf-Hals-Bereiches.

Solid tumors show an altered metabolism with respect to glycolysis in comparison to normal tissue. Recently, the determination of different glycolytic metabolites for tumor diagnosis and therapeutic decision-making became the focus of interest for various research groups. In particular an increased lactate concentration in tumor tissue appears to be a predictor of an adverse prognosis. Imaging of induced bioluminescence in rapidly frozen tumor biopsies is an established technique for the detection of selected substances. In this method the metabolites of interest are biochemically linked to luciferases. A microscopic photon counting system registers the light intensity and after calibration reflects the concentration distribution of metabolites. In contrast to other methods it is possible to detect different metabolites from one specific area of tissue. Preliminary results of a pilot study on oral cancer patients suggest a prognostic impact in terms of high lactate concentrations being associated with poor survival. This technique could increase the validity and significance of tumor grading and might be supportive decision guidance for tumor therapy in the future.